Biomedical subjects

S C Pflugfelder

Publications and source records attributed to S C Pflugfelder.

At least 19 recordsLinked to original sources

PubMed2001

Regulation of MMP-9 production by human corneal epithelial cells.

The matrix metalloproteinases, MMP-2 and MMP-9, are known to be critical extracellular-remodeling enzymes in wound healing and other diseases of the ocular surface. This study investigated the regulation of MMP-2 and MMP-9 in human corneal epithelial cells by growth factors and pro-inflammatory cytokines (IL-1beta and TNF-alpha) they are exposed to, and by doxycycline, a medication used to treat ocular surface disease. Primary human corneal epithelial cell cultures were treated with one of the following cytokines (IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha) or growth factors (EGF, HGF, KGF, PDGF-BB, TGF-alpha, TGF-beta), with or without their corresponding inhibitors. The conditioned media were collected after 24 hr for gelatin zymography and MMP-9 activity assay. Total RNA was extracted from the cells treated for 6 hr and was subjected to RT-PCR and Northern hybridization. Between the two gelatinases, MMP-2 and MMP-9, detected by zymography, the 92 kDa MMP-9 in the conditioned medium was markedly up-regulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. The MMP-9 protein and activity were dose-dependently stimulated by IL-1beta or TNF-alpha at 0.1, 1.0 and 10 ng ml(-1). This up-regulation was nearly abolished by neutralizing antibodies (IL-1beta and TNF-alpha) and by IL-1 receptor antagonist. Semi-quantitative RT-PCR and Northern hybridization disclosed that the MMP-9 transcript was also markedly up-regulated in a dose-dependent manner by IL-1beta and TNF-alpha. Doxycycline (10 microg ml(-1)) suppressed MMP-9 protein level and activity, but not its mRNA, that was stimulated by IL-1beta and TNF-alpha (1 ng ml(-1)). In contrast, the 72 kDa MMP-2 was not significantly modulated by any of these cytokines. In conclusion, production of MMP-9 is stimulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. These factors may play a role in the pathogenesis of MMP-9 mediated corneal matrix degradation. The efficacy of doxycycline in treating ocular surface diseases may be related to its ability to suppress MMP-9 production in the corneal epithelium.

Cell Culture Techniques↗

PubMed2001

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.

Conjunctiva↗

PubMed2001

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.

Administration, Topical↗

PubMed2001

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.

Adult↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Administration, Oral↗

PubMed2001

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.

Amnion↗

PubMed2001

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.

Adult↗

PubMed2000

Increased expression of the type 1 growth factor receptor family in the conjunctival epithelium of patients with keratoconjunctivitis sicca.

PURPOSE: To investigate the expression of type 1 growth factor receptors (epidermal growth factor receptor, ErbB2, and ErbB3) in the conjunctival epithelium of patients with keratoconjunctivitis sicca. METHODS: Immunofluorescent staining and Western blotting were performed to grade the level of expression of the epidermal growth factor receptor ErbB2, and ErbB3 in conjunctival epithelial impression cytologies taken from both eyes of seven normal subjects and 22 patients with keratoconjunctivitis sicca. RESULTS: Epidermal growth factor receptor staining was observed in a greater percentage of keratoconjunctivitis sicca than normal samples (P <.05). ErbB2 and ErB3 staining in the apical conjunctival epithelium was observed in both groups, but stronger ErbB2 and ErbB3 staining was noted in keratoconjunctivitis sicca conjunctival samples (P <.05). The relative levels of expression of these receptor proteins on immunoblots were consistent with immunofluorescent staining. On immunoblots, epidermal growth factor receptor protein was detected in 50% of keratoconjunctivitis sicca samples, but none of the normal samples (P <.025). The expression of ErbB2 and ErbB3 on immunoblots was also greater in the keratoconjunctivitis sicca samples (P <.05). Immunofluorescent staining scores for these receptors were correlated with conjunctival lissamine green staining scores (r =. 574, P <.01 for epidermal growth factor receptor; r =.620, P <.0025 for ErbB2; r =.502, P <.025 for ErbB3) and with corneal fluorescein staining (r =.409, P <.05 for ErbB2; r =.588, P <.005 for ErbB3). CONCLUSION: The expression of the type 1 growth factor receptors is significantly greater in the conjunctival epithelium of eyes with keratoconjunctivitis sicca than normal eyes. The increased expression of these receptors was positively correlated with ocular surface dye staining. The increased expression of these receptors may contribute to the abnormal growth and differentiation of the conjunctival epithelium that occurs in keratoconjunctivitis sicca.

Adult↗

PubMed2000

The effects of experimental tear film removal on corneal surface regularity and barrier function.

PURPOSE: To evaluate corneal surface regularity and asymmetry, corneal thickness, barrier function, and contrast sensitivity after experimental removal of the precorneal tear layer. DESIGN: Prospective, clinic-based, nonrandomized (self-controlled) comparative trial. PARTICIPANTS: Six eyes of six healthy volunteers (three males, three females; age range, 29-40 years). METHODS: A precorneal tear lesion was created by pressing a sterile Biopore (Millipore, Bedford, MA) Teflon membrane against the central cornea. Corneal topography with both the Topographic Modeling System (TMS-1; Computed Anatomy, Tomey Technology, Cambridge, MA) and the Orbscan (Orbscan Inc., Salt Lake City, UT) were performed before the lesion was created and 30 seconds, 1 hour, and 4 hours after the lesion was created. Surface regularity and surface asymmetry indices were evaluated by the TMS-1 topography system. Maximum and minimum keratometric readings, corneal fluorescein staining, contrast sensitivity, and corneal thickness were evaluated before and after the tear lesion. Cytologic membranes were stained for MUC4 mucin using an indirect immunofluorescent staining technique. Confocal microscopy was performed to evaluate the integrity of the corneal epithelium in two eyes. Analysis of variance with polynomial contrasts was used to examine time trends of the outcome variables. MAIN OUTCOME MEASURES: The change in corneal surface regularity and asymmetry indices, corneal thickness, permeability to fluorescein dye, and contrast sensitivity before and after the lesion was made were compared. RESULTS: The corneal epithelium in the area of the lesion showed intense fluorescein staining 30 seconds postlesion but appeared normal by 4 hours. Confluent, homogeneous staining for MUC4 mucin was observed on the membranes used to create the lesion in all cases. The surface regularity index measured with the TMS-1 increased after the lesion was created and decreased toward normal by 4 hours (P = 0.017). Corneal thickness measured by the Orbscan instrument significantly increased in the central (P = 0.001), superior (P = 0.006), inferotemporal (P < 0.001) and superotemporal (P = 0.001) cornea immediately following the lesion and returned to normal by 4 hours. The lesion caused a decrease in visual acuity at 6.30%, 4% and 2.5% contrast sensitivities 1 hour postlesion and these measurements returned to prelesion values by 4 hours (P = 0.085, P = 0.005, P = 0.043). CONCLUSIONS: The precorneal tear layer serves as a permeability barrier and is essential for maintaining a smooth quality optical surface.

Adult↗

PubMed2000

The effects of long-term contact lens wear on corneal thickness, curvature, and surface regularity.

PURPOSE: To evaluate the effect of long-term contact lens wear on corneal thickness, curvature, and surface regularity. DESIGN: A prospective, clinic-based, case-control study. PARTICIPANTS: A total of 40 eyes of 20 normal subjects and 64 eyes of 35 subjects wearing contact lenses for more than 5 years were evaluated. METHODS: The Orbscan Corneal Topography System was used to evaluate the entire corneal thickness and curvature, anterior curvature and the anterior and posterior elevation topographic patterns in normal eyes and subjects wearing contact lenses on a regular basis for more than 5 years. Indices of TMS-1 Corneal Topography System were used to determine corneal surface regularity in subjects wearing contact lenses and normal eyes. All topographic examinations were performed after contact lenses had been removed for at least 2 weeks. MAIN OUTCOME MEASURES: The entire corneal thickness, curvature, surface regularity index (SRI), surface asymmetry index (SAI), potential visual acuity (PVA) and topographic patterns were compared between normal eyes and subjects wearing contact lenses for more than 5 years. RESULTS: The 64 eyes of 35 subjects evaluated by the Orbscan instrument had an average of 13.45 +/- 6.42 years of contact lens wear. The mean corneal thickness in the center and in eight peripheral areas measured in contact lens wearing subjects was significantly reduced by about 30 to 50 microm compared to normal subjects (P < 0.001 for central and peripheral sites). No correlation was noted between central corneal thickness and degree of myopia in diopters (r = 0.15, 0.10 < P < 0.25). The corneal curvature, maximum keratometry (Max K) and minimum keratometry (Min K) readings, were significantly steeper in eyes wearing contact lenses than normal eyes (P < 0.01 for Max K and Min K measured by both instruments). No difference in the mean corneal astigmatism was noted between groups. The SRI and SAI, but not the PVA index, of the TMS-1 were significantly greater in contact lens wearers than in the control group (P < 0.01 for both SRI and SAI, P = 0.15 for PVA). The color-coded patterns of all curvature and elevation maps made with both instruments showed no significant difference between subjects wearing contact lenses and the control group. There was no significant difference between corneal curvature measurements taken with the Orbscan System or the TMS-1 System in both normal and contact lens groups. CONCLUSIONS: Long-term contact lens wear appears to decrease the entire corneal thickness and increase the corneal curvature and surface irregularity.

Adult↗

PubMed2000

Interleukin-6 levels in the conjunctival epithelium of patients with dry eye disease treated with cyclosporine ophthalmic emulsion.

PURPOSE: To evaluate interleukin-6 (IL-6) levels in the conjunctival epithelium of patients with moderate to severe dry eye disease before and after treatment with cyclosporin A ophthalmic emulsion (CsA) or its vehicle. METHODS: Conjunctival cytology specimens were obtained from a subset of patients enrolled in a 6-month randomized, double-masked clinical trial of the efficacy and safety of topical CsA at baseline and after 3 and 6 months of B.I.D. treatment with 0.05% cyclosporine emulsion (n = 13), 0.1% cyclosporine emulsion (n = 8), or vehicle (n = 10). RNA was extracted and a competitive reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the levels of mRNA encoding the inflammatory cytokine IL-6 and a housekeeping gene, G3PDH. Levels of IL-6 and G3PDH were measured and compared. RESULTS: There was no change from baseline in the level of G3PDH after 3 or 6 months in any group. IL-6 normalized for G3PDH (IL-6/G3PDH ratio) was not different from baseline at 3 months but showed a significant decrease from baseline in the group treated with 0.05% CsA (p = 0.048) at 6 months. No significant between-group differences were noted and no correlation was observed between the change in IL-6/G3PDH and corneal fluorescein staining. CONCLUSIONS: This preliminary, small-cohort study showed a decrease in IL-6 in the conjunctival epithelium of moderate to severe dry eye patients treated with 0.05% CsA for 6 months. The observed decrease suggests that dry eye disease involves immune-mediated inflammatory processes that may be decreased by treatment with topical ophthalmic cyclosporine.

Administration, Topical↗

PubMed2000

Recurrent fungal keratitis and endophthalmitis.

PURPOSE: To report a case of recurrent fungal sclerokeratitis and endophthalmitis with a very successful outcome due to aggressive combined surgical and medical therapy. To discuss the management of this potentially devastating infection. METHODS: A 65-year-old man presented with 6 months of left eye redness and irritation after injury from organic matter propelled from an airboat. Initially, he had been treated with foreign body removal, antibiotics, and steroids. He was diagnosed with reactive sclerokeratitis at presentation and was treated with steroids. However, when he did not improve, cultures were obtained and Acremonium species filamentous fungi was identified. Despite treatment with appropriate topical and systemic antifungals, his fungal sclerokeratitis progressed to endophthalmitis. Two therapeutic penetrating keratoplasties (PKs) with iridectomy and intraocular amphotericin B were necessary to eradicate the fungal infection. RESULTS: Visual acuity was restored to 20/25-3 with correction 9 months after initial presentation. There was no recurrence of fungal infection after the second therapeutic PK. CONCLUSION: The possible reasons for recurrence of fungal infection are discussed. The role of timely and aggressive medical and surgical intervention for fungal sclerokeratitis and endophthalmitis in restoring excellent vision is emphasized.

Acremonium↗

PubMed2000

The diagnosis and management of dry eye: a twenty-five-year review.

PURPOSE: To review the advances in the diagnosis, pathogenesis, and management of dry eye disease in the past 25 years. METHODS: Literature review. RESULTS: The preocular tear film is a hydrated mucus gel that contains soluble antimicrobial proteins and growth factors that protect and support the ocular surface. The final common pathway in dry eye is a perturbation of the integrated ocular surface/lacrimal gland reflex unit. Diagnostic tests evaluating tear composition and clearance appear to show stronger correlation with the severity of ocular irritation symptoms and keratoconjunctivitis sicca (KCS) than the conventional Schirmer tests. KCS is a condition of abnormal differentiation and mucus production by the ocular surface epithelium that results in a poorly lubricated, abnormally permeable ocular surface that has increased susceptibility to environmental insults. Chronic subclinical ocular surface inflammation appears to play a key role in the pathogenesis of KCS. New therapeutic strategies are aimed at reducing the ocular surface inflammation of dry eye disease. CONCLUSIONS: There has been a tremendous increase in knowledge regarding dry eye disease in the past 25 years that has resulted in improved diagnostic classification and new targeted therapies.

Animals↗

PubMed2000

Detection of sialomucin complex (MUC4) in human ocular surface epithelium and tear fluid.

PURPOSE: To evaluate human ocular surface epithelium and tear fluid for the presence of sialomucin complex (MUC4), a high-molecular-weight heterodimeric glycoprotein composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis assays were used to identify sialomucin complex RNA in ocular surface epithelia. Immunoprecipitation and immunoblot analysis were used to identify immunoreactive species in human tears and in the corneal and conjunctival epithelia using antibodies specific for carbohydrate and peptide epitopes on the sialomucin complex subunits. Immunofluorescence staining was used to detect sialomucin complex in frozen sections and impression cytology specimens of human cornea and conjunctival epithelia. RESULTS: ASGP-1- and ASGP-2-specific sequences were amplified from RNA extracted from both conjunctival and corneal epithelial biopsies by RT-PCR. Sialomucin complex transcripts were also detected in these tissues by Northern blot analysis, with a greater level of RNA detected in the peripheral than the central corneal epithelium. Sialomucin complex was immunoprecipitated from tear fluid samples and both corneal and conjunctival epithelia and detected by immunoblot analysis with specific anti-ASGP-1 and anti-ASGP-2 antibodies. The ASGP-1 peptide antibody HA-1 stained the full thickness of the corneal and conjunctival epithelia. In contrast, antibody 15H10, which reacts against a carbohydrate epitope on ASGP-1, stained only the superficial epithelial layers of these tissues. No staining was observed in the conjunctival goblet cells. CONCLUSIONS: Sialomucin complex was originally identified in rat mammary adenocarcinoma cells and has recently been shown to be produced by the ocular surface epithelia of rats. Furthermore, it has been identified as the rat homologue of human MUC4 mucin. The present studies show that it is expressed in the stratified epithelium covering the surface of the human eye and is present in human tear fluid. Expression of a carbohydrate-dependent epitope on the mucin subunit (ASGP-1) of sialomucin complex occurs in a differentiation-dependent fashion. Sialomucin complex joins MUC1 as another membrane mucin produced by the human ocular surface epithelia but is also found in the tear fluid, presumably in a soluble form, as found on the rat ocular surface.

Antibody Specificity↗

PubMed2000

Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant.

PURPOSE: To evaluate human corneal epithelial culture supernatant and tear fluid for the presence of activators and inhibitors of matrix metalloproteinase (MMP)-9, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1, respectively, and to evaluate the effect of MMP-3 on the activation of MMP-9 in these specimens. METHODS: Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system. RESULTS: MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants. CONCLUSIONS: MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.

Blotting, Western↗

PubMed2000

Doxycycline inhibition of interleukin-1 in the corneal epithelium.

PURPOSE: To evaluate the effect of doxycycline on the regulation of interleukin (IL)-1 expression and activity in human cultured corneal epithelium. METHODS: Human corneal limbal epithelium (HLE) was cultured from explants prepared from limbal rings of donor corneas. Primary cultured limbal epithelial cells were treated with either 10 microg/ml lipopolysaccharide (LPS), LPS with 10 microg/ml doxycycline, or LPS with 0.1 mg/ml methylprednisolone (MP) for 24 hours. The intracellular and supernatant protein amounts of IL-1alpha, the precursor and mature forms of IL-1beta, IL-1 receptor antagonist (IL-1 RA), and the intracellular level of IL-1beta-converting enzyme (ICE) were measured with enzyme-linked immunosorbent assays (ELISAs). Western blot analysis was performed to evaluate IL-1 RA protein. mRNA steady state amounts were determined by RNase protection assay (RPA) for IL-1alpha, IL-1beta, IL-1 RA, and ICE. RESULTS: LPS increased the mRNA and protein amounts of intracellular and released IL-1alpha, mature IL-1beta, and IL-1 RA. Doxycycline inhibited the LPS-induced IL-1beta increase in the mRNA and protein amounts in the corneal epithelium and upregulated the expression of the anti-inflammatory IL-1 RA protein. In addition, doxycycline reduced the steady state level of the cellular ICE protein but did not affect the level of ICE transcripts. IL-1beta secreted to the conditioned media of HLE was functionally active in inducing matrix metalloproteinase (MMP)-1 and MMP-3 in cultured corneal fibroblasts. Doxycycline significantly decreased IL-1beta bioactivity in the supernatants from LPS-treated corneal epithelial cultures. These effects were comparable to those induced by the corticosteroid, MP. CONCLUSIONS: Doxycycline can suppress the steady state amounts of mRNA and protein of IL-beta and decrease the bioactivity of this major inflammatory cytokine. These data may partially explain the clinically observed anti-inflammatory properties of doxycycline. The observation that doxycycline was equally potent as a corticosteroid, combined with the relative absence of adverse effects, makes it a potent drug for a wide spectrum of ocular surface inflammatory diseases.

Anti-Bacterial Agents↗

PubMed2000

[The effect of artificial tears on corneal surface regularity in dry eye].

OBJECTIVE: To evaluate the regularity of corneal surface and the effect of artificial tears on the corneal surface regularity in dry eye. METHODS: The surface regularity index (SRI), surface asymmetry index (SAI), the indices of TMS-1 corneal topography system, and potential visual acuity (PVA) were used to investigate the corneal regularity and corneal vision in 33 normal subjects (64 eyes) and 22 dry eye patients (42 eyes) before and after instillation of artificial tears. RESULTS: The SRI and SAI were significantly elevated and the PVA was remarkably decreased in patients with dry eye compared to that of normal subjects (P < 0.001). The average amount of astigmatism was also significantly increased in dry eye compared to that in normal eyes (P = 0.02). In the patients with dry eye, the SRI and SAI were positively correlated with corneal fluorescein staining scores. The SRI, SAI and astigmatism were significantly reduced, the PVA was improved and the irregular topographic pattern was markedly decreased in the dry eyes after the usage of artificial tears. CONCLUSION: The dry eye has an irregular corneal surface and the indices of TMS-1, SRI and SAI can be used to objectively diagnose this disorder, evaluate the severity of the dry eye and the effect of artificial tears on it. Artificial tears can smooth the corneal surface and improve the visual acuity in patients with dry eye.

Aged↗