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Biomedical subjects

Stephen C Pflugfelder

Publications and source records attributed to Stephen C Pflugfelder.

At least 37 records · Page 2Linked to original sources
PubMed2005

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.

Animals↗
PubMed2005

Pathways of corneal and ocular surface inflammation: a perspective from the cullen symposium.

The goal of this symposium was to coalesce information presented by 22 investigators in the field of corneal and ocular surface inflammation into common pathways of inflammation. The perspective elucidated in this article defines the components of the normal ocular surface immune architecture and describes the consensus reached on the mechanisms/pathways involved in 1) acute inflammation; 2) late-stage (chronic) response; and 3) allergic disease. Seven diagrams didactically illustrate mechanisms. This paper is the introductory article in a supplement containing 18 articles by the symposium participants.

Journal Article↗
PubMed2005

Stress-activated protein kinase signaling pathways in dry eye and ocular surface disease.

Inflammation is recognized as a key component in the pathogenesis of dry eye and a variety of ocular surface diseases. Stress-activated protein kinases have been identified as pathways signaling ocular surface stresses, such as increased tear film osmolarity and ultraviolet light exposure. Activation of these stress pathways results in transcription of stress related genes, including inflammatory cytokines (e.g. interleukin-1 and tumor necrosis factor (TNF)-alpha and matrix metalloproteinases (MMPs), such as MMP-9. Treatment of osmo-stressed cultured corneal epithelia with inhibitors of the stress associated kinase c-jun N-terminal kinases was found to decrease production of MMPs by these cells. These findings suggest that SAPKs may be key therapeutic targets for dry eye and ocular surface diseases.

Journal Article↗
PubMed2005

Evaluation of ocular surface inflammation in the presence of dry eye and allergic conjunctival disease.

The ocular inflammatory diseases dry eye and allergic conjunctivitis are mediated by CD4+ T cells. Th1 cells secrete interferon (IFN)-gamma and are implicated in mediating the disease process in dry eye. Allergic conjunctivitis has been classically defined as a Th2 disease because of the predominance of Th2 cytokines interleukin (IL)-4 and IL-13. A multi-hit antigen challenge mouse model of allergic conjunctivitis provides evidence that IFN-gamma, a Th1 cytokine, acts as an endothelium gatekeeper by regulating endothelial expression of vascular adhesion molecule-1 required for inflammatory conjunctival cell infiltration. Current research encourages an in-depth evaluation of the exact role Th1 and Th2 cells play in ocular inflammation.

Journal Article↗
PubMed2005

Matrix metalloproteinases in corneal inflammation.

Matrix metalloproteinases (MMPs) are a family of calcium-dependent, zinc-containing endoproteinases capable of degrading the extracellular matrix and basement membrane. Many inflammatory stimuli, such as wound healing, ultraviolet, hyperosmolarity, and stress, can stimulate production of inflammatory cytokines and a variety of MMPs, including gelatinases, collagenases, and stromelysins. Pro-inflammatory cytokines and growth factors can further promote the production of MMPs. Thus, MMPs participate in and promote the inflammation process. MMP-9 may be a most important amplifying factor for corneal inflammation. Mitogen activated protein kinases are involved in MMPs amplified inflammation. These pathway studies provide new therapeutic potential to block MMP mediated inflammation.

Journal Article↗
PubMed2005

Effect of experimental dry eye on tear sodium concentration in the mouse.

PURPOSE: To examine the changes in tear volume, sodium concentration, and osmolarity in BALB/c and C57BL/6J mice in response to experimental dry eye (EDE). METHODS: Tear volumes were determined at baseline and after 2 days of EDE using phenol red-impregnated cotton thread. Tear fluid was collected by instilling 1 microl of distilled water into the conjunctival sac and collecting the diluted tear fluid in a 0.5-microL glass capillary tube. CoroNa Red in dimethyl sulfoxide DMSO was added, and the fluorescent intensity was measured, which was compared to a standard curve of sodium concentrations. The resulting concentration was then corrected by the proper dilution factor. The tear osmolarity owing to sodium was calculated by comparing the calculated tear sodium concentration to a standard curve established by a vapor pressure osmometer. Serum osmolarity was also determined using a vapor pressure osmometer. RESULTS: After 2 days of EDE, tear volume significantly decreased from 0.093 microL to 0.028 microL in BALB/c mice and from 0.066 microL to 0.026 microL in C57BL/6J mice. There was a concomitant significant increase in tear osmolarity from 177 mOsm/L to 300 mOsm/L in C57BL/6J mice. Tear osmolarity nearly doubled from 285 mOsm/L to 559 mOsm/L in BALB/C mice with EDE, approaching statistical significance (P=0.12). No change in the serum osmolarity was observed in either mouse strain. CONCLUSIONS: These experiments show the ability of these new techniques for determining tear volume and estimating tear osmolarity in mice. By mimicking the findings of human dry eye disease, these findings validate the relevance of the mouse model for studying the pathogenesis of human keratoconjunctivitis sicca.

Animals↗
PubMed2005

Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface.

PURPOSE: To investigate whether hyperosmolar stress stimulates production of inflammatory mediators and activates the mitogen-activated protein kinase (MAPK) signaling pathways, c-jun n-terminal kinases (JNKs), extracellular-regulated kinases (ERKs), and p38 on the mouse ocular surface. METHODS: 129SvEv/CD-1 mixed mice were treated with a balanced salt solution (BSS) (305 mOsM) or a hyperosmotic saline solution (HOSS) (500 mOsM). Untreated age-matched mice were used as controls. The concentrations of interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) were measured by enzyme-linked immunosorbent assay. Gelatinase activity was determined by in situ zymography. Corneal and conjunctival epithelia were lysed for Western blot with MAPK antibodies or used for semiquantitative reverse transcription and polymerase chain reaction and gene array. RESULTS: Compared with age-matched controls and mice treated with BSS, the concentration of IL-1beta in tear fluid washings and the concentrations of IL-1beta and TNF-alpha and gelatinolytic activity in the corneal and conjunctival epithelia were significantly increased in mice treated with HOSS for 2 days. The expressions of IL-1beta, TNF-alpha, and matrix metalloproteinase 9 (MMP-9) messenger RNA by the corneal and conjunctival epithelia were also notably stimulated in mice treated with HOSS. The levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia were slightly increased in mice treated with BSS, but markedly increased in mice treated with HOSS. CONCLUSIONS: These results show that the hyperosmolarity stimulates expression and production of IL-1beta, TNF-alpha, and MMP-9 and activates JNK, ERK, and p38 MAPK signaling pathways on the mouse ocular surface. These findings suggest that hyperosmolar stress, as it may occur in dry eye, promotes ocular surface inflammation.

Animals↗
PubMed2005

Topical cyclosporine inhibits conjunctival epithelial apoptosis in experimental murine keratoconjunctivitis sicca.

PURPOSE: Increased apoptosis in the conjunctival epithelium has been observed in experimental murine keratoconjunctivitis sicca (KCS). Topical cyclosporine (CsA) has been noted to reduce conjunctival epithelial apoptosis in chronic canine and human KCS. The purpose of this study is to determine if topical CsA treatment inhibits conjunctival epithelial apoptosis in a murine model of KCS. METHODS: Dry eye was induced in 3 groups of C57BL6 mice by subcutaneous injection of scopolamine TID and exposure to an air draft and low-humidity environment for 16 hours per day for 12 days. The dry eye control group received no topical treatment; a second group received 1 microL of 0.05% CsA topically TID (dry eye + CsA); and the third group received 1 microL of the castor oil vehicle of CsA topically TID (dry eye + vehicle). Normal mice were used as untreated controls. After 12 days, the mice were killed, and the right eyes and eyelids were excised, frozen, and cryosectioned. Transmission electron microscopy (TEM) was performed on conjunctival and corneal samples taken from the left eyes. Apoptosis was detected in frozen sections with the ApopTag (ISOL) In Situ Oligo Ligation Kit, which specifically detects DNA fragmentation. Immunohistochemical staining was performed to detect activated caspase-3. Conjunctival goblet cell number was counted in tissue sections stained with period acid Schiff (PAS) reagent. These assays were performed on 2 separate sets of mice. RESULTS: Compared with untreated controls and dry eye mice receiving CsA, the number of ISOL-positive epithelial cells in the bulbar and tarsal conjunctiva was significantly greater in the dry eye control and dry eye mice + vehicle groups (P < 0.01 for both groups). There was no significant difference in the number of ISOL-positive conjunctival epithelial cells between the dry eye control and dry eye + vehicle mice. There was no significant difference in ISOL-positive cells in the corneal epithelium between the untreated controls and the 3 treatment groups. Dry eye + CsA mice showed less activated caspase-3 staining than the dry eye control and the dry eye + vehicle groups. TEM showed loss of superficial differentiated cells and extensive nuclear fragmentation characteristic of apoptosis in the dry eye control and dry eye + vehicle groups but not in the dry eye + CsA group. There was significant loss of goblet cells in the bulbar and tarsal conjunctivae of the dry eye control and the dry eye + vehicle groups compared with untreated controls and the dry eye + CsA group. CONCLUSIONS: Topical CsA significantly reduced conjunctival epithelial apoptosis and protected against goblet cell loss in experimental murine KCS. Inhibition of apoptosis appears to be a key mechanism for the therapeutic effect of CsA for KCS.

Administration, Topical↗
PubMed2005

Therapy of progressive rheumatoid arthritis-associated corneal ulceration with infliximab.

PURPOSE: To review the relative efficacy of the TNFalpha antagonist infliximab in treating 3 patients with rheumatoid arthritis-associated peripheral ulcerative keratitis. METHODS: Review of the clinical course of 3 nonconsecutive patients with progressive rheumatoid arthritis-associated peripheral ulcerative keratitis who were initially treated with conventional immunosuppressant therapy and subsequently treated with infliximab. RESULTS: All 3 patients experienced progressive corneal ulceration on oral prednisone and weekly oral or intramuscular methotrexate. They all demonstrated a marked reduction in conjunctival injection, closure of their corneal epithelial defects, and arrest of corneal thinning after receiving infliximab. Two out of 3 patients required patch grafts for treatment of corneal perforations before starting infliximab, and they had no further keratolysis after receiving this agent. CONCLUSIONS: Infliximab was effective in arresting progressive rheumatoid arthritis-associated peripheral ulcerative keratitis that was refractory to conventional immunomodulatory therapy.

Aged↗
PubMed2005

Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells.

PURPOSE: To evaluate the effects of TGF-beta1 and doxycycline on production of gelatinase MMP-9 and activation of Smad, c-Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways in human corneal epithelial cells. METHODS: Primary human corneal epithelial cells were cultured to confluence. The cells were treated with different concentrations of TGF-beta1 (0.1, 1, or 10 ng/mL), with or without TGF-beta1-neutralizing mAb (5 microg/mL), SP600125 (30 microM), PD98059 (40 microM), SB202190 (20 microM), or doxycycline (5-40 microg/mL) for different lengths of time. Conditioned media were collected from cultures treated for 24 to 48 hours to evaluate the MMP-9 production by zymography and activity assay. Total RNA was isolated from cells treated for 6 to 24 hours to evaluate MMP-9 expression by semiquantitative RT-PCR and Northern hybridization. Cells treated for 5 to 60 minutes were lysed in RIPA buffer for Western blot with phospho-specific antibodies against Smad2, JNK1/2, ERK1/2, or p38. RESULTS: TGF-beta1 increased expression, production, and activity of MMP-9 by human corneal epithelial cells in a concentration-dependent fashion. TGF-beta1 also induced activation of Smad2, JNK1/2, ERK1/2, and p38 within 5 to 15 minutes, with peak activation at 15 to 60 minutes. Doxycycline markedly inhibited the TGF-beta1-induced production of MMP-9 and activation of the Smad, JNK1/2, ERK1/2, and p38 signaling pathways. Its inhibitory effects were of a magnitude similar to SP600125, PD98059, and SB202190, specific inhibitors of the JNK1/2, ERK1/2, and p38 pathways, respectively. CONCLUSIONS: These findings demonstrated that doxycycline inhibits TGF-beta1-induced MMP-9 production and activity, perhaps through the Smad and MAPK signaling pathways. These inhibitory effects may explain the reported efficacy of doxycycline in treating MMP-9-mediated ocular surface diseases.

Anti-Bacterial Agents↗
PubMed2005

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 DeltaNp63 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 DeltaNp63 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.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2004

Antiinflammatory therapy for dry eye.

PURPOSE: To present evidence establishing the relationship between inflammation and dry eye and supporting the use of antiinflammatory therapy for dry eye. DESIGN: Analysis of literature. METHODS: Research studies that evaluated inflammation in dry eye pathogenesis and clinical trials of antiinflammatory therapies for dry eye were reviewed. RESULTS: There is increasing evidence that decreased tear secretion, decreased tear turnover, and desiccation promote inflammation on the ocular surface. An increase in soluble mediators (cytokines and proteases) in the tear fluid, adhesion molecule expression by the conjunctival epithelium, and T-cell infiltration of the conjunctiva have been observed in dry eye patients. This inflammation appears to have a role in the pathogenesis of the ocular surface epithelial disease, termed keratoconjunctivitis sicca (KCS), that develops in dry eye. Clinical improvement of KCS has been observed after therapy with antiinflammatory agents including corticosteroids, cyclosporin and doxycycline. Cyclosporin A emulsion was approved by the Food and Drug Administration as therapy for dry eye. Randomized placebo-controlled FDA clinical trials showed that cyclosporine A was superior to vehicle in stimulating aqueous tear production, decreasing corneal punctuate fluorescein staining, reducing symptoms of blurred vision, and decreasing artificial tear use in patients with KCS. No ocular or systemic toxicity was observed from this medication. CONCLUSIONS: Ocular surface and lacrimal gland inflammation has been identified in dry eye that plays a role in the pathogenesis of KCS. Antiinflammatory therapy has efficacy for treating KCS. Cyclosporin A is the first FDA approved therapy for this indication. It improved signs and symptoms of KCS, and it is safe for long-term use.

Anti-Inflammatory Agents↗
PubMed2004

A randomized, double-masked, placebo-controlled, multicenter comparison of loteprednol etabonate ophthalmic suspension, 0.5%, and placebo for treatment of keratoconjunctivitis sicca in patients with delayed tear clearance.

PURPOSE: To evaluate loteprednol etabonate ophthalmic 0.5% suspension, versus placebo for treatment of the inflammatory component of keratoconjunctivitis sicca in patients with delayed tear clearance. DESIGN: Randomized, double-masked, placebo-controlled clinical trial. METHODS: Sixty-four patients with keratoconjunctivitis sicca and delayed tear clearance were randomly assigned to receive either loteprednol or vehicle 4 times a day for 4 weeks. Patients were evaluated at weeks 2 and 4 of treatment and 2 weeks after treatment was discontinued. Symptoms were scored using a visual analog scale (VAS) of 1 to 100. Corneal fluorescein staining was scored 0 to 4 in five areas. Conjunctival injection was graded 0 to 3 in the inferior bulbar, nasal bulbar, and inferior tarsal areas. Lid margin injection was graded 0 to 3. Safety was assessed by funduscopy, lens examination, biomicroscopy, visual acuity, and Goldmann tonometry, and by monitoring adverse events and changes in symptoms. RESULTS: In subsets of patients with at least moderate clinical inflammation, there was a significant difference between the loteprednol-treated group and vehicle-treated group after 2 weeks of therapy. The differences did not reach statistical significance at 4 weeks, although the loteprednol-treated patients retained their improvement compared with the vehicle-treated group. Safety evaluations showed both treatments to be well tolerated and similar in the frequency and type of adverse event reported. CONCLUSION: The use of topical loteprednol etabonate 0.5% 4 times a day may be beneficial in patients who have keratoconjunctivitis sicca with at least a moderate inflammatory component.

Androstadienes↗
PubMed2004

Tear clearance implications for ocular surface health.

Tear clearance/turnover provides a global assessment of the function of the lacrimal functional unit and of tear exchange on the ocular surface. It is an indirect measure of dry eye induced inflammation on the ocular surface. It shows better correlation with the severity of ocular irritation symptoms and corneal epithelial disease in dry eye than the Schirmer 1 test. Delayed tear clearance may prove to be the best measure for identifying patients with tear film disorders who may respond to anti-inflammatory therapy.

Diagnostic Techniques, Ophthalmological↗
PubMed2004

The role of the lacrimal functional unit in the pathophysiology of dry eye.

The majority of dry eye symptoms are due to a chronic inflammation of the lacrimal functional unit resulting in a loss of tear film integrity and normal function. This leads to a reduction in the ability of the ocular surface to respond to environmental challenges. The underlying cause of tear film dysfunction is the alteration of tear aqueous, mucin, and lipid components. This may result from a systemic autoimmune disease or a local autoimmune event. A lack of systemic androgen support to the lacrimal gland has been shown to be a facilitative factor in the initiation of this type of pathophysiology. Tear secretion is controlled by the lacrimal functional unit consisting of the ocular surface (cornea, conjunctiva, accessory lacrimal glands, and meibomian glands), the main lacrimal gland and the interconnecting innervation. If any portion of this functional unit is compromised, lacrimal gland support to the ocular surface is impeded. Factors such as neurogenic inflammation and T cell involvement in the disease pathogenesis as well as newly developed animal models of ocular surface inflammation are discussed.

Androgens↗
PubMed2004

Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures.

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.

Adolescent↗
PubMed2004

TGF-beta1 stimulates production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) by human corneal epithelial cells.

Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of ocular surface diseases. This study investigated the regulated expression of gelatinases (MMP-2 and -9), collagenases (MMP-1 and -13) and stromelysins (MMP-3, -10, and -11) by TGF-beta1 in cultured human corneal epithelial cells. Primary human corneal epithelial cell cultures were grown to confluence and treated with different concentrations (0.1, 1.0, 10 ng ml(-1)) of TGF-beta1 in serum-free medium for 6-24 hr. Total RNA was isolated from cultured cells with or without TGF-beta1 treatment for 6 hr and subjected to semi-quantitative RT-PCR and Northern hybridization. Conditioned media were collected from cultures with or without TGF-beta1 treatment for 24 hr to evaluate the MMP production by ELISA and activity assays. Semi-quantitative RT-PCR revealed that the expressions of MMP-9, -1, -13, -3, -10 and -11 mRNA were up-regulated by TGF-beta1 in a concentration-dependent fashion, while MMP-2 and MMP-14 production did not change. Northern hybridization confirmed these findings. Gelatin zymography, MMP ELISA and activity assays showed concentration-dependent stimulated production and activity of MMP-9, -1, -13, -3 and -10 protein in the conditioned media of cultures treated for 24 hr with TGF-beta1. In conclusion, our results demonstrate that TGF-beta1 stimulates the expression and production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) in human corneal epithelial cells. These findings suggest that TGF-beta1 may play a role in the pathogenesis of MMP mediated ocular surface diseases, such as sterile corneal ulceration.

Cells, Cultured↗