Systemic anti-inflammatory therapy aided by double-headed nanoparticles in a canine model of acute intraocular inflammation.

Novel approaches circumventing blood-ocular barriers in systemic drug delivery are lacking. We hypothesize receptor-mediated delivery of curcumin (CUR) across intestinal and ocular barriers leads to decreased inflammation in a model of lens-induced uveitis. CUR was encapsulated in double-headed polyester nanoparticles using gambogic acid (GA)-coupled polylactide-co-glycolide (PLGA). Orally administered PLGA-GA2-CUR led to notable aqueous humor CUR levels and was dosed (10 mg/kg twice daily) to adult male beagles (n = 8 eyes) with induced ocular inflammation. Eyes were evaluated using a semiquantitative preclinical ocular toxicology scoring (SPOTS) and compared to commercial anti-inflammatory treatment (oral carprofen 2.2 mg/kg twice daily) (n = 8) and untreated controls (n = 8). PLGA-GA2-CUR offered improved protection compared with untreated controls and similar protection compared with carprofen, with reduced aqueous flare, miosis, and chemosis in the acute phase (<4 hours). This study highlights the potential of PLGA-GA2 nanoparticles for systemic drug delivery across ocular barriers.

A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.

PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin ß1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.

Age-related spontaneous lacrimal keratoconjunctivitis is accompanied by dysfunctional T regulatory cells.

In both humans and animal models, the development of Sjögren syndrome (SS) and non-SS keratoconjunctivitis sicca (KCS) increases with age. Here, we investigated the ocular surface and lacrimal gland (LG) phenotype of NOD.B10.H2b mice at 7-14, 45-50, and 96-100 weeks. Aged mice develop increased corneal permeability, CD4+ T-cell infiltration, and conjunctival goblet cell loss. Aged mice have LG atrophy with increased lymphocyte infiltration and inflammatory cytokine levels. An increase in the frequency of CD4+Foxp3+ T regulatory cells (Tregs) was observed with age in the cervical lymph node (CLN), spleen, and LG. These CD4+CD25+ cells lose suppressive ability, while maintaining expression of Foxp3 (forkhead box P3) and producing interleukin-17 (IL-17) and interferon-γ (IFN-γ). An increase of Foxp3+IL-17+ or Foxp3+IFN-γ+ cells was observed in the LG and LG-draining CLN. In adoptive transfer experiments, recipients of either purified Tregs or purified T effector cells from aged donors developed lacrimal keratoconjunctivitis, whereas recipients of young Tregs or young T effector cells failed to develop disease. Overall, these results suggest inflammatory cytokine-producing CD4+Foxp3+ cells participate in the pathogenesis of age-related ocular surface disease.

CD8⁺ cells regulate the T helper-17 response in an experimental murine model of Sjögren syndrome.

This study investigated the regulatory function of CD8⁺ cells in T helper-17 (Th17) cell-mediated corneal epithelial barrier disruption that develops in a murine desiccating stress (DS) model that resembles Sjögren syndrome. CD8⁺ cell depletion promoted generation of interleukin-17A (IL-17A)-producing CD4⁺ T cells via activation of dendritic cells in both the ocular surface and draining cervical lymph nodes in C57BL/6 mice subjected to DS. T-cell-deficient nude recipient mice receiving adoptively transferred CD4⁺ T cells from CD8⁺ cell-depleted donors exposed to DS displayed increased CD4⁺ T-cell infiltration and elevated IL-17A and CC-chemokine attractant ligand 20 levels in the ocular surface, which was associated with greater corneal barrier disruption. Enhanced DS-specific corneal barrier disruption in CD8-depleted donor mice correlated with a Th17-mediated expression of matrix metalloproteinases (MMP-3 and MMP-9) in the recipient corneal epithelium. Co-transfer of CD8⁺CD103⁺ regulatory T cells did not affect the ability of DS-specific pathogenic CD4⁺ T cells to infiltrate and cause ocular surface disease in the nude recipients, showing that CD8⁺ cells regulate the efferent arm of DS-induced immune response. In summary, CD8⁺ regulatory cells suppress generation of a pathogenic Th17 response that has a pivotal role in DS-induced disruption of corneal barrier function.

Potential autocrine regulation of interleukin-33/ST2 signaling of dendritic cells in allergic inflammation.

This study identified a novel phenomenon that dendritic cells (DCs) produced interleukin (IL)-33 via Toll-like receptor (TLR)-mediated innate pathway. Mouse bone marrow-derived DCs were treated with or without microbial pathogens or recombinant murine IL-33. IL-33 mRNA and protein were found to be expressed by DCs and largely induced by several microbial pathogens, highly by lipopolysaccharide (LPS) and flagellin. Using two mouse models of topical challenge by LPS and flagellin and experimental allergic conjunctivitis, IL-33-producing DCs were observed in ocular mucosal surface and the draining cervical lymph nodes in vivo. The increased expression levels of myeloid differentiation primary-response protein 88 (MyD88), nuclear factor (NF)-κB1, NF-κB2, and RelA accompanied by NF-κB p65 nuclear translocation were observed in DCs exposed to flagellin. IL-33 induction by flagellin was significantly blocked by TLR5 antibody or NF-κB inhibitor quinazoline and diminished in DCs from MyD88 knockout mice. IL-33 stimulated the expression of DC maturation markers, CD40 and CD80, and proallergic cytokines and chemokines, OX40L, IL-4, IL-5, IL-13, CCL17 (C-C motif chemokine ligand 17), TNF-α (tumor necrosis factor-α), and IL-1ß. This stimulatory effect of IL-33 in DCs was significantly blocked by ST2 antibody or soluble ST2. Our findings demonstrate that DCs produce IL-33 via TLR/NF-κB signaling pathways, suggesting a molecular mechanism by which local allergic inflammatory response may be amplified by DC-produced IL-33 through potential autocrine regulation.

Low humidity environmental challenge causes barrier disruption and cornification of the mouse corneal epithelium via a c-jun N-terminal kinase 2 (JNK2) pathway.

Patients with tear dysfunction often experience increased irritation symptoms when subjected to drafty and/or low humidity environmental conditions. The purpose of this study was to investigate the effects of low humidity stress (LHS) on corneal barrier function and expression of cornified envelope (CE) precursor proteins in the epithelium of C57BL/6 and c-jun N-terminal kinase 2 (JNK2) knockout (KO) mice. LHS was induced in both strains by exposure to an air draft for 15 (LHS15D) or 30 days (LHS30D) at a relative humidity <30%RH. Nonstressed (NS) mice were used as controls. Oregon-green-dextran uptake was used to measure corneal barrier function. Levels of small proline-rich protein (SPRR)-2, involucrin, occludin, and MMP-9 were evaluated by immunofluorescent staining in cornea sections. Wholemount corneas immunostained for occludin were used to measure mean apical cell area. Gelatinase activity was evaluated by in situ zymography. Expression of MMP, CE and inflammatory cytokine genes was evaluated by qPCR. C57BL/6 mice exposed to LHS15D showed corneal barrier dysfunction, decreased apical corneal epithelial cell area, higher MMP-9 expression and gelatinase activity and increased involucrin and SPRR-2 immunoreactivity in the corneal epithelium compared to NS mice. JNK2KO mice were resistant to LHS-induced corneal barrier disruption. MMP-3,-9,-13, IL-1α, IL-1ß, involucrin and SPRR-2a RNA transcripts were significantly increased in C57BL/6 mice at LHS15D, while no change was noted in JNK2KO mice. LHS is capable of altering corneal barrier function, promoting pathologic alteration of the TJ complex and stimulating production of CE proteins by the corneal epithelium. Activation of the JNK2 signaling pathway contributes to corneal epithelial barrier disruption in LHS.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13.

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK- populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Autoimmunity at the ocular surface: pathogenesis and regulation.

A healthy ocular surface environment is essential to preserve visual function, and as such the eye has evolved a complex network of mechanisms to maintain homeostasis. Fundamental to the health of the ocular surface is the immune system, designed to respond rapidly to environmental and microbial insults, whereas maintaining tolerance to self-antigens and commensal microbes. To this end, activation of the innate and adaptive immune response is tightly regulated to limit bystander tissue damage. However, aberrant activation of the immune system can result in autoimmunity to self-antigens localized to the ocular surface and associated tissues. Environmental, microbial and endogenous stress, antigen localization, and genetic factors provide the triggers underlying the immunological events that shape the outcome of the diverse spectrum of autoimmune-based ocular surface disorders.

Spontaneous T cell mediated keratoconjunctivitis in Aire-deficient mice.

BACKGROUND/AIMS: Patients with autoimmune polyendocrinopathy-candiasis-ectodermal dystrophy (APECED) develop severe keratoconjunctivitis, corneal scarring and visual loss, but the precise pathogenesis is unknown. This study evaluated the ocular surface immune cell environment, conjunctival goblet cell density and response to desiccating environmental stress of the autoimmune regulatory (Aire) gene knockout murine model of APECED. METHODS: Aire-deficient and wild type (WT) mice were subjected to desiccating stress from a drafty, low-humidity environment and pharmacological inhibition of tear secretion for 5 days. Immune cell populations (CD4(+), CD8(+), CD11b(+), CD45(+)) and goblet cell density were measured in ocular surface tissues and meibomian glands, and compared with baseline values. RESULTS: Greater CD4(+) T cell populations were observed in the conjunctival epithelium of Aire-deficient mice (p<0.001) compared with WT. Aire-deficient mice also had greater numbers of CD4(+), CD8(+), and CD11b(+) cells in the peripheral cornea at baseline and following desiccating stress. The meibomian glands of Aire-deficient mice demonstrated greater CD4(+), CD8(+), CD45(+) and CD11b(+) cells at baseline (p<0.001) and following desiccating stress. Conjunctival goblet cell density was lower at baseline and following desiccating stress in Aire-deficient compared with WT mice (p<0.001). CONCLUSION: Aire-deficiency leads to infiltration of CD4(+) and CD8(+) T cells on the ocular surface and meibomian glands, which is accompanied by goblet cell loss. Desiccating stress promotes this proinflammatory milieu. Immune-mediated mechanisms play a role in the severe blepharitis and keratoconjunctivitis in the murine model of APECED.

IL-17 disrupts corneal barrier following desiccating stress.

T helper (Th)-17 is a recently identified subtype of Th response that has been implicated in host defense and autoimmunity. We investigated whether there is evidence for a Th-17 response in human and experimental murine dry eye (DE). Gene expression in the human DE conjunctiva showed increased levels of the Th-17 inducers, interleukin (IL)-23, IL-17A, and interferon-gamma (IFN-gamma). In the murine model, we found that desiccating stress increased matrix metalloproteinase-9, Th-17-associated genes (IL-6, IL-23, transforming growth factor-beta1 and -2, IL-23R, IL-17R, IL-17A, retinoid-related orphan receptor-gammat, and CC chemokine attractant ligand-20) and IFN-gamma in cornea and conjunctiva. Furthermore, we found a significantly increased concentration of IL-17 in tears and number of IL-17-producing cells on the ocular surface. Antibody neutralization of IL-17 ameliorated experimental DE-induced corneal epithelial barrier dysfunction and decreased the expression of matrix metalloproteinases 3 and 9. Taken together, these findings suggest that IL-17 has a role in corneal epithelial barrier disruption in DE.

Rationale for anti-inflammatory therapy in dry eye syndrome: [review] / Bases da terapia antiinflamatória em síndrome do olho seco: [revisão]

Dry eye is a multifactorial condition that results in a dysfunctional lacrimal functional unit. Evidence suggests that inflammation is involved in the pathogenesis of the disease. Changes in tear composition including increased cytokines, chemokines, metalloproteinases and the number of T cells in the conjunctiva are found in dry eye patients and in animal models. This inflammation is responsible in part for the irritation symptoms, ocular surface epithelial disease, and altered corneal epithelial barrier function in dry eye. There are several anti-inflammatory therapies for dry eye that target one or more of the inflammatory mediators/pathways that have been identified and are discussed in detail.

Olho seco é uma doença multifatorial que resulta em disfunção da unidade lacrimal glandular. Evidências sugerem que inflamação está involvida na patogênese da doença. Mudanças na composição das lágrimas, incluindo aumento de citocinas, quimiocinas, metaloproteinases e o número de células T na conjuntiva são encontrados em pacientes com olho seco e em modelos animais. Esta inflamação é responsável em parte pelos sintomas de irritação, doença epitelial de surperfície ocular e função epitelial de barreira alterada em olho seco. Existem várias terapias antiinflamatórias que se direcionam para um ou mais mediadores/vias que foram identificados e são discutidos em detalhe.

Expression of glial cell-derived neurotrophic factor and its receptor in the stem-cell-containing human limbal epithelium.

AIM: To evaluate the expression pattern of glial cell line-derived neurotrophic factor (GDNF) with its receptors GDNF family receptor alpha-1 (GFR alpha-1) and Ret in the human corneal and limbal tissues, as well as in the primary human limbal epithelial cultures (PHLEC). METHODS: Expression of GDNF and its receptors, and the co-localisation with stem cell associated and differentiation markers were evaluated by immunofluorescent staining, western blot analysis and real-time PCR in the fresh human corneoscleral tissues, as well as in the PHLEC. Single cell colony-forming and wound-healing assays were also evaluated in PHLEC. RESULTS: GDNF and GFR alpha-1 were found to be expressed by a subset of basal cells and co-localised with ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2) and p63, but not with cytokeratin 3 in the human limbal basal epithelium. In PHLEC, they were expressed by a small population of cells in the less differentiated stage. The GDNF and GFR alpha-1-positive subpopulations were enriched for the expression of ABCG2 and p63 (p<0.01). Recombinant human GDNF promoted the proliferation and wound healing of epithelial cells in the PHLEC. In contrast, Ret was abundantly located in the human corneal epithelium except for the basal cells of the limbal epithelium. CONCLUSION: These findings indicate that GDNF and GFR alpha-1 may represent a property for the phenotype of human corneal epithelial precursor cells. GDNF may signal independently of Ret through GFR alpha-1 in the stem cell-containing limbal epithelium.

Rationale for anti-inflammatory therapy in dry eye syndrome.

Dry eye is a multifactorial condition that results in a dysfunctional lacrimal functional unit. Evidence suggests that inflammation is involved in the pathogenesis of the disease. Changes in tear composition including increased cytokines, chemokines, metalloproteinases and the number of T cells in the conjunctiva are found in dry eye patients and in animal models. This inflammation is responsible in part for the irritation symptoms, ocular surface epithelial disease, and altered corneal epithelial barrier function in dry eye. There are several anti-inflammatory therapies for dry eye that target one or more of the inflammatory mediators/pathways that have been identified and are discussed in detail.

Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease.

PURPOSE: To compare the expression of the pro- and anti-inflammatory forms of interleukin (IL)-1 in the tear fluid and conjunctival epithelium of normal eyes and those with dry-eye disease. METHODS: The concentrations of IL-1 alpha, IL-1 beta (precursor and mature forms), and IL-1 receptor antagonist (IL-1Ra) were measured by ELISA in tear fluid samples obtained from normal individuals and patients with dry eye who had rosacea-associated meibomian gland disease (MGD) or Sjögren's syndrome (SS) aqueous tear deficiency (ATD). These cytokines were also measured in normal tear fluid before and after nasal stimulation to induce reflex tearing. The relative expression of these cytokines was evaluated in conjunctival impression cytology specimens and conjunctival biopsy tissue obtained from normal subjects and SS ATD-affected patients using immunofluorescent staining. Matrix metalloproteinase (MMP)-9 concentration and activity in the tear fluid were evaluated with gelatin zymography and with an MMP-9 activity assay kit, respectively. RESULTS: Compared with normal subjects, the concentration of IL-1 alpha and mature IL-1 beta in the tear fluid was increased, and the concentration of precursor IL-1 beta was decreased in patients with MGD (P < 0.05, P = 0.02, and P < 0.01, respectively) and SS ATD (P < 0.001, P = 0.02, and P < 0.001, respectively). There was no significant change in the concentration of IL-1 alpha, precursor IL-1 beta, and IL-1Ra in reflex tear fluid, indicating that the lacrimal glands may secrete these cytokines. The activity of MMP-9, a physiological activator of IL-1 beta, was significantly elevated in the tear fluid of both dry-eye groups compared with normal subjects. A strong positive correlation was observed between the intensity of corneal fluorescein staining and the tear fluid IL-1 alpha concentration (r(2) = 0.17, P < 0.02) and the mature-to-precursor IL-1 beta ratio (r(2) = 0.46, P < 0.001). Positive immunofluorescent staining for IL-1 alpha, mature IL-1 beta, and IL-1Ra was observed in a significantly greater percentage of conjunctival cytology specimens from eyes with SS ATD than in those from normal eyes (P < 0.01 for IL-1 alpha, P < 0.009 for mature IL-1 beta, and P < 0.05 for IL-1Ra). CONCLUSIONS: Dry-eye disease is accompanied by an increase in the proinflammatory forms of IL-1 (IL-1 alpha and mature IL-1 beta) and a decrease in the biologically inactive precursor IL-1 beta in tear fluid. Increased protease activity on the ocular surface may be one mechanism by which precursor IL-1 beta is cleaved to the mature, biologically active form. The conjunctival epithelium appears to be one source of the increased concentration of IL-1 in the tear fluid of patients with dry-eye disease. These results suggest that IL-1 may play a key role in the pathogenesis of keratoconjunctivitis sicca.

Pseudokeratoconus caused by rosacea.

PURPOSE: A case of inferior corneal thinning and high astigmatism with features of keratoconus in a patient with long-standing ocular rosacea is described. METHODS: Axial curvature mapping was performed with the Tomey TMS-1 videokeratoscopy instrument and corneal thickness mapping was performed with the Orbscan Corneal Topography System (CTS). Tear clearance was assessed by measuring the concentration of fluorescein in the tear fluid with a fluorometer. RESULTS: There were inferior corneal thinning and opacification in both eyes. Tear fluorescein clearance was markedly delayed in the right eye. There was asymmetric inferior corneal steepening in both eyes with I-S values of 1 in the right eye and 5.9 in the left eye. There were 5.9 diopters of astigmatism at 85 degrees in the right eye and 7.3 diopters of astigmatism at 73 degrees in the left eye. Corneal pachymetry mapping with the Orbscan CTS showed a normal central corneal thickness and maximal thinning in the inferotemporal periphery of the right cornea and inferonasal periphery of the left cornea. CONCLUSION: Chronic ocular rosacea can produce inferior corneal thinning and high astigmatism with some features of keratoconus. The inferior pattern of thinning in rosacea may be related to chronic exposure of the inferior cornea to inflammatory and matrix-degrading factors in the inferior tear meniscus.

Treatment of recalcitrant recurrent corneal erosions with inhibitors of matrix metalloproteinase-9, doxycycline and corticosteroids.

PURPOSE: To review the efficacy of inhibitors of matrix metalloproteinase-9, corticosteroids, and doxycycline for treatment of recalcitrant recurrent corneal erosion. METHODS: Retrospective, clinic-based, interventional case series. The medical records of seven consecutive patients who were treated between January 1995 to January 2000 for recurrent corneal erosion who had not responded to conventional therapy were reviewed. Treatment of seven eyes of seven patients consisted of oral doxycycline (50 mg, two times a day) for 2 months along with a topical corticosteroid (either methylprednisolone 1%, prednisolone acetate 1%, or fluoromethalone 0.1%) three times a day, for 2 to 3 weeks. The effects of doxycycline and methylprednisolone on metalloproteinase-9 activity in human corneal epithelial cultures were evaluated by gelatin zymography and a commercial metalloproteinase-9 activity assay kit. RESULT: Fingernail injury in three of the seven eyes was the most common form of corneal injury. There was no evidence of epithelial basement membrane or corneal stromal dystrophy in any of the patients, although epithelial microcysts were observed in the involved area in three patients. One eye had intact elevated corneal epithelium that showed abnormal diffuse staining with fluorescein dye, and six eyes had a corneal epithelial defect at the time of presentation. In all seven eyes, pain resolved and epithelial defects healed within 2 to 10 days after initiation of therapy. No recurrence was observed during an average follow-up period of 21.9 months (range, 1.5 to 60 months). Methylprednisolone and doxycycline each produced a statistically significant decrease in amount and activity of metalloproteinase-9 in conditioned media of human corneal epithelial cultures. CONCLUSIONS: Therapy with a combination of medications that inhibit metalloproteinase-9 produced rapid resolution and prevented further recurrence of cases of recurrent corneal erosions that were unresponsive to conventional therapies.

Effects of laser in situ keratomileusis on tear production, clearance, and the ocular surface.

PURPOSE: To evaluate components of the integrated ocular surface/lacrimal gland unit in a series of patients before and after undergoing bilateral laser in situ keratomileusis (LASIK). DESIGN: Prospective, noncomparative case series. PARTICIPANTS: Forty-eight eyes of 14 men and 34 women (age range, 26-54; mean, 39.2 years) who underwent bilateral LASIK for myopia or myopic astigmatism. METHODS: LASIK was performed using a VISX Star Excimer Laser (Santa Clara, CA). Patients completed a questionnaire containing 11 questions that evaluated the character and severity of ocular irritation symptoms. Snellen visual acuity, tear fluorescein clearance, corneal fluorescein staining, aqueous tear production by the Schirmer 1 test, and corneal and conjunctival sensitivity were measured in each eye. Corneal surface regularity (SRI) was evaluated with the Tomey TMS-1 (Tomey, Cambridge, MA) topography instrument. Each randomly chosen eye was evaluated 1 to 2 days (T0) before LASIK and 7 days (T1), 1 (T2), 2 (T3), 6 (T4), 12 (T5), and 16 (T6) months postoperatively. A Wilcoxon test, two-tailed paired t test, Friedman test, or analysis of variance were used for statistical comparisons. MAIN OUTCOME MEASURES: Components of the integrated ocular surface/lacrimal gland unit. RESULTS: Both corneal and conjunctival sensitivity were noted to be significantly decreased from preoperative levels at 1week, 1 month, 12 months, and 16 months postoperatively (P < 0.0002 at each time point). Symptom severity scores were significantly increased at 1 week, 12 months, and 16 months postoperatively (P < 0.007 at all time points). The mean Schirmer 1 test scores were 24 +/- 14 mm preoperatively, and they decreased to 18 +/- 14 mm by 1 month postoperatively (P < 0.001). Tear fluorescein clearance showed a linear increase postoperatively and was significantly greater than baseline (P < 0.001) at each time point. There was a significant increase in punctate corneal fluorescein staining at 1 week postoperatively (P < 0.0001), but staining returned to baseline by 12 months. There was a statistically significant increase in SRI 1 week postoperatively (P < 0.007) with return to baseline levels by 6 months. CONCLUSIONS: Sensory denervation of the ocular surface after bilateral LASIK disrupts ocular surface tear dynamics and causes irritation symptoms. Patients undergoing LASIK should be informed of these risks.

Suppression of interleukin 1alpha and interleukin 1beta in human limbal epithelial cells cultured on the amniotic membrane stromal matrix.

AIMS: Amniotic membrane (AM) transplantation reduces inflammation in a variety of ocular surface disorders. The aim of this study was to determine if AM stroma suppresses the expression of the IL-1 gene family in cultured human corneal limbal epithelial cells. METHODS: Human corneal limbal epithelial cells were cultured from limbocorneal explants of donor eyes on plastic or on the AM stroma. Transcript expression of IL-1alpha, IL-1beta, IL-1 receptor antagonist (RA), and GAPDH was compared with or without addition of lipopolysaccharide to their serum-free media for 24 hours using RNAse protection assay (RPA). Their protein production in the supernatant was analysed by ELISA. RESULTS: Expression of IL-1alpha and IL-1beta transcripts and proteins was significantly reduced by cells cultured on the AM stromal matrix compared with plastic cultures whether lipopolysaccharide was added or not. Moreover, expression of IL-1 RA by cells cultured in the lipopolysaccharide-free medium was upregulated by AM stromal matrix. The ratio between IL-1 RA and IL-1alpha protein levels in AM cultures was higher than in plastic cultures. CONCLUSIONS: AM stromal matrix markedly suppresses lipopolysaccharide induced upregulation of both IL-1alpha and IL-1beta. These data may explain in part the effect of AM transplantation in reducing ocular surface inflammation, underscoring the unique feature of the AM as a substrate for tissue engineering.

The effect of punctal occlusion on tear production, tear clearance, and ocular surface sensation in normal subjects.

PURPOSE: To evaluate the effect of temporary punctal occlusion on tear production, tear clearance, and ocular surface sensation in normal subjects. METHODS: Noncomparative interventional case series. Punctal occlusion with silicone punctal plugs was performed on nine normal subjects without complaints of ocular irritation and no known history of ocular surface disease. The lower punctum of both eyes was occluded in five subjects. The upper and lower puncta of only one eye were occluded in four subjects. Corneal and conjunctival sensations were measured with the Cochet-Bonnet anesthesiometer. Tear fluorescein clearance was evaluated with a CytoFluor II fluorophotometer by measuring the fluorescein concentration in minimally stimulated tear samples collected from the inferior tear meniscus 15 minutes after instillation of fluorescein. Schirmer test was performed without anesthesia. The tests were performed at days 0, 1, 3, 7, and 14 to 17 after punctal occlusion. Relationships were analyzed with linear regressions, and a quadratic term was used to model a return to preocclusion levels. Paired t test was used to study the change in tear fluorescein concentration. RESULTS: In subjects who had the lower puncta of both eyes occluded, conjunctival sensation decreased in both eyes (right eye, P =.008; left eye, P =.003), but there was no change in corneal sensation. Their tear fluorescein clearance did not show a significant change from baseline (P =.90). However, a decrease in Schirmer test scores approached statistical significance (P =.056). In subjects with both puncta of only one eye occluded, we noted a decrease in corneal sensation (occluded eye, P =.042; nonoccluded eye, P =.036), conjunctival sensation (occluded, P =.001; nonoccluded, P =.060), and Schirmer scores (occluded, P =.022; nonoccluded, P =.011). Linear regression did not show a significant change in tear fluorescein clearance for either eye (occluded, P =.28; nonoccluded, P =.44). However, paired t test showed a significant worsening of tear clearance in the occluded eye from day 0 to day 3 (P =.001) followed by a subsequent improvement in tear clearance from day 3 to the end of the study period (P =.045). Paired t test did not reveal any significant changes in tear clearance in the nonoccluded eye. The quadratic term of the linear regression model demonstrated an increase toward preocclusion levels that approached statistical significance for corneal sensation (occluded, P =.053; nonoccluded, P =.099). It was statistically significant for conjunctival sensation (occluded, P =.001; nonoccluded, P =.045) and Schirmer scores (occluded, P =.047; nonoccluded, P =.044). CONCLUSIONS: Temporary punctal occlusion in normal subjects decreases tear production and ocular surface sensation. Our findings suggest that in addition to blocking tear drainage, punctal occlusion may affect the ocular surface/lacrimal gland interaction. These effects were more pronounced in subjects with both upper and lower puncta occluded. In normal subjects, there appears to be an autoregulatory mechanism to return tear production, tear clearance, and ocular surface sensation to preocclusion levels 14 to 17 days after punctal occlusion.

Expression of the receptor tyrosine kinases, epidermal growth factor receptor, ErbB2, and ErbB3, in human ocular surface epithelia.

PURPOSE: To investigate the distribution and relative level of expression of the receptor tyrosine kinases, epidermal growth factor receptor (EGFR), ErbB2 and ErbB3, in human ocular surface epithelia. METHODS: Immunofluorescent staining was performed to identify expression of the EGFR, ErbB2 and ErbB3 in the corneal, limbal and conjunctival epithelium in tissue sections and impression cytologies taken from normal human eyes. Western blotting was undertaken to confirm the results of immunofluorescent staining. RESULTS: The three receptor tyrosine kinases, EGFR, ErbB2 and ErbB3, were detected in human corneal, limbal and conjunctival epithelia by immunofluorescent staining. Strong staining for the EGFR was observed in the basal epithelial cells at all 3 sites and throughout the corneal epithelium. Minimal or no staining for the EGFR was observed in the superficial conjunctival and limbal epithelia. The strongest staining for ErbB2 and ErbB3 was observed in the superficial ocular surface epithelium. All three receptors were detected in the corneal, limbal and conjunctival epithelium by western blot. CONCLUSION: EGFR, ErbB2 and ErbB3 are expressed by the ocular surface epithelia. EGFR is preferentially expressed by the basal epithelial cells that have the greatest proliferative potential. In contrast, ErbB2 and ErbB3 are preferentially expressed by the superficial differentiated ocular surface epithelia.