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Haque R.M.,Inspire Pharmaceuticals | Torkildsen G.L.,ORA Inc | Brubaker K.,Inspire Pharmaceuticals | Zink R.C.,Inspire Pharmaceuticals | And 3 more authors.
Cornea | Year: 2010

Purpose: To evaluate the effect of 4 weeks of treatment with azithromycin ophthalmic solution 1% on eyelid bacterial load, tear cytokines, and signs and symptoms of blepharitis. Methods: Twenty-six subjects (mean age 64.2 years; 65% female; 100% white) with moderate to severe blepharitis received azithromycin ophthalmic solution 1% in the absence of warm compresses or eyelid scrubs for 28 days (twice a day on days 1 and 2 and once a day on days 3-28). Blepharitis signs and symptoms were evaluated at baseline (day 1) and compared with end of treatment (day 29) and 2 follow-up visits (2 and 4 weeks posttreatment). Tear collection and eyelid margin bacterial cultures were performed at baseline and end of treatment. Tear cytokines were measured by a multiplex immunobead assay. Results: Four-week azithromycin treatment demonstrated significant decreases from baseline in investigator-rated signs of meibomian gland plugging, eyelid margin redness, palpebral conjunctival redness, and ocular discharge (P ≤ 0.002) at day 29, which persisted 4 weeks posttreatment (P ≤ 0.006). Subject-reported symptoms of eyelid itching, foreign body sensation/sandiness/ grittiness, ocular dryness, ocular burning/pain, and swollen/heavy eyelids also demonstrated significant improvement from baseline (P < 0.001 for all symptoms and time points, except P = 0.037 for ocular dryness at visit 4). Eyelid margin culture exhibited significant decreases in coagulase-negative staphylococci and Corynebacterium xerosis bacteria. Changes in tear cytokine concentrations were not observed. Twelve subjects experienced 19 adverse events, 15 of which were ocular and none of which were serious. Conclusion: Azithromycin provided significant improvement in signs and symptoms of blepharitis after 4 weeks of treatment compared with baseline and persisted in the 4-week follow-up period. © 2010 by Lippincott Williams and Wilkins.


Ma P.,Sun Yat Sen University | Ma P.,Cullen Eye Institute | Wang Z.,Sun Yat Sen University | Pflugfelder S.C.,Cullen Eye Institute | Li D.-Q.,Cullen Eye Institute
Experimental Eye Research | Year: 2010

Human peptidoglycan recognition proteins (PGLYRPs) are a novel family of pattern recognition receptors, and also act as anti-bacterial proteins. This study was to explore the toll-like receptor (TLR)-mediated regulation of PGLYRPs in human corneal epithelial cells (HCECs). Fresh human donor corneoscleral tissues were used to prepare cryosections. Primary HCECs, established from limbal explants, were treated with microbial ligands to TLRs 1-9 for 4-48 h, with or without pretreatment of TLR antibodies, NFkB inhibitor, or siRNA transfection. The mRNA of PGLYRPs was evaluated by RT and real-time PCR, and their proteins and NFkB activation were determined by immunostaining and Western blot. The nuclear IRF3 activity was quantified using an ELISA-based TransAM kit. PGLYRP-2, -3 and -4 were found to be expressed by human corneal epithelium while PGLYRP-1 was not detected. In primary HCEC cultures, PGLYRP-3 and -4 were constitutively expressed while PGLYRP-2 was largely inducible. PGLYRP-2 was induced by bacterial components, Pam3CSK4, PGN, flagellin and FSL-1, ligands for TLR2/1, 2, 5 and 2/6, respectively. Interestingly, PGLYRP-2 was strongest stimulated by polyI:C representing viral dsRNA. TLR3 antibody or NFkB inhibitor blocked IRF3 and NFkB p65 activation as well as polyI:C-stimulated PGLYRP-2. RNA interference indicates that the polyI:C-induced PGLYRP-2 was dramatically blocked in the cells transfected with siRNA-TRIF but neither siRNA-MyD88 nor the negative control siRNA-F. These findings suggest that human corneal epithelium may response to viral or bacterial infection by producing PGLYRPs through TLRs, and the induction of PGLYRP-2 by dsRNA was through TLR3-TRIF-IRF3-NFkB signaling pathways. © 2009 Elsevier Ltd. All rights reserved.


Zheng X.,Cullen Eye Institute | Zheng X.,Shanxi Eye Hospital | Ma P.,Cullen Eye Institute | Ma P.,Shandong University | And 5 more authors.
Investigative Ophthalmology and Visual Science | Year: 2010

Purpose. To explore the potential role of thymic stromal lym-phopoietin (TSLP) and its downstream molecules in the development of ocular allergic inflammation using a short ragweed (SRW)-induced mouse model of allergic conjunctivitis (AC). Methods. BALB/c mice were topically challenged with SRW pollen after they were sensitized with SRW in the footpad. After the last SRW challenge, the corneal epithelium, conjunctiva, and cervical lymph nodes were harvested for total RNA extraction and gene expression by RT and real-time PCR, and whole eye globes were collected to make cryosections for immunohistochemical staining. Results. Repeated topical challenges with SRW allergen generated typical signs of AC in mice. Compared with the untreated controls, TSLP mRNA expression and immunoreactivity were significantly increased in the corneal and conjunctival epithelia of SRW-induced AC mice. CD11c+ and OX40L+ immunoreactive cells largely infiltrated the conjunctiva with increased mRNA levels of CD11c, TSLPR, and OX40L detected in the corneal epithelium, conjunctiva, and cervical lymph nodes. CD4+ Th2 cell infiltration was evidenced by increased levels of mRNA and immunoreactivity of CD4, IL-4, IL-5, and IL-13 in the ocular surface, mainly in the conjunctiva, accompanied by increased expression of OX40, STAT6, and GATA3, in AC mice. The maturation of immature DCs was observed with the use of TSLP containing conditioned media from corneal epithelial cultures exposed to polyI:C, which stimulates TSLP production. Conclusions. This study provides new findings regarding the role of local mucosal epithelial cells in the initiation of ocular allergic inflammation by producing a novel proallergic cytokine, TSLP, which activates dendritic cells to prime Th2 differentiation and allergic inflammation through the TSLP-TSLPR and OX40L-OX40 signaling pathway. © Association for Research in Vision and Ophthalmology.


Zheng X.,Cullen Eye Institute | Zheng X.,Shanxi Eye Hospital | de Paiva C.S.,Cullen Eye Institute | Li D.-Q.,Cullen Eye Institute | And 2 more authors.
Investigative Ophthalmology and Visual Science | Year: 2010

Purpose. To explore the phenomenon that corneal and conjunctival tissues subjected to desiccating stress (DS) promote Th17 differentiation by stimulating the production of Th17-inducing cytokines through a dendritic cell (DC)-mediated pathway. Methods. Experimental dry eye was created by subjecting C57BL/6 mice to desiccating environmental stress. Corneal and conjunctival explants from dry eye or control mice were cocul-tured with DCs for 24 hours before CD4+ T cells were added for an additional 4 to 7 days. Expression of Th17-associated genes in the cornea, conjunctiva, DCs, and CD4+ T cells was evaluated by real-time PCR. Cytokine concentrations in cocul-ture supernatants were measured by immunobead assay. IL-17-producing T cells were identified by ELISPOT bioassay. Results. Higher levels of IL-17A, TGF-j31, TGF-j32, IL-6, IL-23, and IL-1 j3 mRNA transcripts and TGF-/31, IL-6, and IL-1/3 protein were observed in corneal epithelium and conjunctiva from dry eye mice. DCs cocultured with epithelial explants from dry eye mice for 2 days produced higher levels of TGF-/31, IL-6, IL-23, and IL-1/3 mRNA transcripts and of TGF-/31, IL-6, and IL-1/3 protein. CD4+ T cells cocultured with DCs and epithelial explants from dry eye mice expressed increased levels of IL-17A, IL-17F, IL-22, CCL-20, and retinoic acid receptor-related orphan receptor-yt mRNA transcripts and increased IL-17A protein and number of IL-17-producing T cells (Th17 cells). Conclusions. These findings demonstrate that DS creates an environment on the ocular surface that stimulates the production of Th17-inducing cytokines by corneal and conjunctival epithelia that promote Th17 differentiation through a dendritic cell-mediated pathway. © Association for Research in Vision and Ophthalmology.


Zhang X.,Cullen Eye Institute | Zhang X.,Wenzhou Medical College | Chen W.,Cullen Eye Institute | Chen W.,Wenzhou Medical College | And 8 more authors.
American Journal of Pathology | Year: 2011

We investigated the role of CD4 + T-cellproduced interferon (IFN)-γ on corneal epithelial apoptosis in a murine desiccating stress (DS) model that resembles Sjögren's syndrome. The DS model was generated in C57BL/6 (B6) and B6 IFN-γknockout (B6γKO) mice. Adoptive transfer of CD4 + T cells from DS-exposed donor to recombination activating gene (RAG)-1 -/- recipient mice and topical neutralization of IFN-γ were performed to determine whether IFN-γ produced by pathogenic CD4 + T cells promotes corneal epithelial apoptosis. Apoptosis in corneal epithelia was assessed by evaluating the expression and activity of caspases 3, 8, and 9. The activation of caspase-8 mediated increased corneal epithelial apoptosis in B6 mice after DS, and this was exacerbated by subconjunctival IFN-γ injection. B6γKO mice were resistant to DS-induced apoptosis; however, B6γKO mice receiving IFN-γ developed apoptosis similar to that observed in B6 wild-type mice. Adoptive transfer of CD4 + T cells from donors subjected to DS increased corneal epithelial apoptosis via activation of caspase-8 in recipients, similar to that in the donor mice. Topical neutralization of IFN-γ in adoptive transfer recipients decreased corneal epithelial apoptosis. DS, IFN-γ administration, or CD4 + T-cell adoptive transfer had no effect on the expression and activation of the intrinsic apoptosis mediator, caspase-9. CD4 + T-cellproduced IFN-γ plays a pivotal role in DS-induced corneal epithelial apoptosis via activation of the extrinsic apoptotic pathway. © 2011 American Society for Investigative Pathology.


Zhang X.,Cullen Eye Institute | Zhang X.,Shenyang He Eye Hospital | Schaumburg C.S.,Allergan, Inc. | Coursey T.G.,Cullen Eye Institute | And 9 more authors.
Mucosal Immunology | Year: 2014

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. © 2014 Society for Mucosal Immunology.


Henriksson J.T.,University of Houston | De Paiva C.S.,Cullen Eye Institute | Farley W.,Cullen Eye Institute | Pflugfelder S.C.,Cullen Eye Institute | And 2 more authors.
Cornea | Year: 2013

PURPOSE:: To investigate the normal palpebral conjunctival histology in C57BL/6 mice and the structural changes that occur in a dry eye model. METHODS:: Twenty-four male and female C57BL/6 mice, 8 untreated and 16 exposed to experimental ocular surface desiccating stress (DS). Ocular dryness was induced by administration of scopolamine hydrobromide (0.5 mg/0.2 mL) four times a day for 5 days (DS5) or 10 days (DS10). Counts and measurements were obtained using anatomical reference points, and goblet cell density was investigated with a variety of stains. RESULTS:: Near the junction between the lid margin and the normal palpebral conjunctiva, the epithelium had an average thickness of 45.6 ± 10.5 μm, 8.8 ± 2.0 cell layers, versus 37.7 ± 5.6 μm, 7.4 ± 1.3 layers in DS10 (P < 0.05). In the goblet cell-populated palpebral region, the normal epithelium was thicker (P < 0.05) than on DS5 and DS10. In the control, 43% of the goblet cells were covered by squamous epithelium compared with 58% (DS5) and 63% (DS10) (P < 0.05). A decreased number of periodic acid-Schiff (PAS)-stained goblet cells and Alcian blue-stained goblet cells were observed in the dry eye. Not all goblet cells were stained with PAS and Alcian blue. CONCLUSION:: The mouse palpebral conjunctival epithelium was structurally similar to the human. After DS, the palpebral conjunctival epithelium decreased in thickness and goblet cell access to the surface seemed to be inhibited by surrounding epithelial cells, potentially slowing down their migration to the surface. Differential staining with PAS and Alcian blue suggests that there may be different subtypes of conjunctival goblet cells. Copyright © 2012 by Lippincott Williams Wilkins.


McClellan A.J.,Cullen Eye Institute | Volpe E.A.,Cullen Eye Institute | Zhang X.,Cullen Eye Institute | Zhang X.,Shenyang He Eye Hospital | And 4 more authors.
American Journal of Pathology | Year: 2014

Dry eye in humans displays increased prevalence in the aged and in women. Here, we investigated the ocular surfaces and lacrimal glands of aged mice of both sexes. We surveyed three different ages [young, middle-aged (6 to 9 months), and elderly] by investigating severity markers of dry eye disease (DED). We observed an age-dependent dry eye phenotype as early as 6 to 9 months: increased corneal surface irregularity, increased corneal barrier disruption, conjunctival CD4+ T-cell infiltration, and loss of mucin-filled goblet cells. Expression of interferon-γ, IL-17 mRNA transcripts was increased in the conjunctiva and IL-17A, matrix metallopeptidase 9, and chemokine ligand 20 in the corneas of elderly mice. Elderly male mice develop more of a skewed response of type 1 T helper cell, whereas female mice have a bias toward type 17 T helper cell in the conjunctiva. In the lacrimal gland, an increase in CD4+ and CD8+ T cells and B cells and a decrease in activated dendritic cells were observed. Adoptive transfer of CD4+ T cells isolated from elderly mice transferred DED into young immunodeficient recipients, which was more pronounced from male donors. Our findings show the development of DED in aging mice. Pathogenic CD4+ T cells that develop with aging are capable of transferring DED from older mice to naive immunodeficient recipients. Taken together, our results indicate that age-related autoimmunity contributes to development of DED with aging. Copyright © 2014 American Society for Investigative Pathology.


Pflugfelder S.C.,Cullen Eye Institute | Stern M.E.,Cullen Eye Institute | Stern M.E.,DuPont Company
Expert Review of Clinical Immunology | Year: 2014

The 4th Cullen Symposium, held April 17 and 18, 2014, included expert researchers in mucosal immunity of the eye and other mucosal surfaces, particularly the gut. The theme of the meeting was environmental sensing mechanisms in mucosal tissues and their relevance for initiating ocular surface inflammation in dry eye. There are a number of shared features between the ocular surface and other mucosal surfaces, but distinct differences may exist in the type and distribution of mucins and microbiota. Mechanisms to regulate DC maturation and prevent tissue-damaging inflammation are shared among these sites. Epithelial and dendritic cells are key environmental sensors participating in initiation of innate and adaptive immune responses in response to a variety of environmental stresses. © 2014 Informa UK Ltd.


De Paiva C.S.,Cullen Eye Institute | Raince J.K.,Cullen Eye Institute | McClellan A.J.,Cullen Eye Institute | Shanmugam K.P.,Cullen Eye Institute | And 7 more authors.
Mucosal Immunology | Year: 2011

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. © 2011 Society for Mucosal Immunology.

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