Dagoberto Monroy

Correlation of Goblet Cell Density and Mucosal Epithelial Membrane Mucin Expression with Rose Bengal Staining in Patients with Ocular Irritation

PURPOSE This study was designed to compare goblet cell densities and mucosal epithelial membrane mucin (MEM) expression in impression cytology specimens obtained from control subjects and patients with one of the following clinically defined diseases: aqueous tear deficiency (ATD) associated with Sjögren syndrome, ATD not associated with Sjögren syndrome, inflammatory Meibomian gland disease associated with rosacea, and Meibomian gland atrophy. These data were correlated with ocular surface rose Bengal staining scores, Schirmer scores, and HLA-DR antigen staining of conjunctival epithelial cells. METHODS Goblet cell density and MEM expression were studied by impression imprints with immunohistochemical staining using an anti-mucosal epithelial membrane mucin antibody in the temporal and inferior bulbar and inferior tarsal conjunctiva of study subjects. RESULTS Goblet cell density adjacent to the temporal limbus was significantly reduced at 3 mm posterior to the temporal limbus in both aqueous tear deficiency groups compared with the other groups and in patients with Sjögren syndrome compared with all other groups. In the inferior tarsus, goblet cell density was significantly reduced in patients with non-Sjögren syndrome ATD as compared with all other groups, except those with inflammatory Meibomian gland disease. Mucosal epithelial membrane mucin expression in the bulbar and tarsal conjunctiva was absent in a greater percentage of patients with Sjögren syndrome compared with all other groups. Total ocular surface rose Bengal staining scores were significantly higher in patients with Sjögren syndrome as compared with all other groups and in patients with non-Sjögren syndrome ATD as compared with control groups. Rose Bengal staining scores and Schirmer I test results (without anesthesia) were inversely correlated with bulbar, but not tarsal, conjunctival goblet cell densities, and with the absence of bulbar conjunctival MEM expression. CONCLUSIONS These results suggest that reduced goblet cell density and mucosal epithelial cell mucin expression could explain increased rose Bengal staining in patients with aqueous tear deficiency. In addition, MEM may be regarded as a marker for normal differentiation of ocular surface epithelia, with its absence signifying the development of squamous metaplasia.

Correlation of tear fluorescein clearance and Schirmer test scores with ocular Irritation symptoms

OBJECTIVE To correlate and compare the Schirmer 1 test and a new method of measuring tear fluorescein clearance with the CytoFluor II fluorometer with the severity of ocular irritation symptoms, clinical signs of meibomian gland disease, corneal fluorescein staining scores, and corneal and conjunctival sensitivity. DESIGN Case-control study. PARTICIPANTS Forty patients presenting with a chief complaint of ocular irritation, and 40 asymptomatic control subjects of similar age distribution. INTERVENTION All subjects completed a symptom questionnaire, a baseline ocular examination, fluorescein clearance test (FCT), and Schirmer 1 test. MAIN OUTCOME MEASURES The FCT was performed 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 5 microl of 2% sodium fluorescein. Severity of ocular irritation was assessed with a symptom questionnaire. Schirmer 1 test, biomicroscopic meibomian gland evaluation, corneal fluorescein staining score, and corneal and conjunctival sensation scores were assessed with the Cachet-Bonnet anesthesiometer in all subjects. RESULTS Irritation symptoms correlated with higher log tear fluorescein concentration (symptomatic 3.08 +/- 0.62 units/,microl, normal control 1.89 +/- 0.7 units/microl, P < 0.005) and lower Schirmer 1 test scores (symptomatic 12.6 mm, normal control 22.3 mm, P < 0.005). The FCT showed greater predictive value for identifying ocular irritation than the Schirmer 1 test. A fluorescein concentration of 274 units//microl eliminated 80% of the normal subjects (specificity) and identified 85% of the abnormal subjects (sensitivity). Log of tear fluorescein concentration and the Schirmer 1 test correlated with meibomian gland orifice metaplasia (2.81 +/- 0.78 units/microl and 14.47 +/- 9.53 mm in those with metaplasia vs. 1.83 +/- 0.71 units/microl and 23.14 +/- 7.67 mm in those without metaplasia, P < 0.001) and with the percentage of acinar dropout. Both log of tear fluorescein concentration and the Schirmer 1 test correlated with corneal fluorescein staining (Pearson correlation of 0.394 P < 0.0001 for Schirmer 1 test and 0.312 P < 0.005 for log of tear fluorescein). In addition, log of tear fluorescein and Schirmer 1 test scores correlated with corneal and conjunctival sensation scores (Spearmans rho for corneal sensation: log of tear fluorescein -0.38, P < 0.003, Schirmer 1 test -0.39, P < 0.002, and for conjunctival sensation: log of tear fluorescein -0.391, P < 0.001, Schirmer 1 test -0.23, P < 0.061). CONCLUSIONS The FCT shows a greater predictive value for ocular irritation than the Schirmer 1 test. It correlates better with age, meibomian gland dysfunction, and decreased corneal and conjunctival sensation. Decreased tear clearance was identified as a risk factor for ocular irritation, even in subjects with normal Schirmer scores. This simple technique may provide new clues into the mechanism and therapy of ocular irritation.

Inflammatory Cytokines in the Tears of Patients with Ocular Rosacea

OBJECTIVE The purpose of the study is to compare tear fluid concentrations of interleukin-1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), and epidermal growth factor (EGF) in ocular rosacea with those in control subjects and to examine the relation between tear functions, such as production and clearance rate, and the concentrations of cytokines in tear fluid. PARTICIPANTS AND INTERVENTION Fourteen patients with severe meibomian gland disease, facial rosacea, and symptoms of ocular irritation were examined for ocular surface disease, tear production, and tear clearance rate (TCR). Twelve control subjects, frequency-matched for age, and 15 ideal normal subjects with no ocular symptoms and normal tear function were assessed using the same parameters. Minimally stimulated tear samples (20 microl) were drawn from each subject and analyzed using a sandwich enzyme-linked immunosorbent assay to detect IL-1alpha, TNF-alpha, and EGF. RESULTS Tear IL-1alpha concentration was significantly higher in patients with rosacea than in age-matched (P = 0.003) and ideal control subjects (P < 0.001). Tumor necrosis factor-alpha was not detected in patients or control subjects, indicating levels of less than 10 pg/ml. Epidermal growth factor was not significantly higher in patients with rosacea than in age-matched control subjects. Tear turnover LN(TCR) was lower in patients with rosacea than in both age-matched (P = 0.048) and ideal control subjects (P = 0.002). Schirmer I scores were statistically lower in patients with rosacea than in ideal control subjects (P = 0.013), but not age-matched control subjects. Interleukin-1alpha was correlated inversely with LN(TCR) (r= -0.58, P < 0.0001) and Schirmer I (r = -0.39, P = 0.012). CONCLUSIONS Concentrations of IL-1alpha are present in normal tears but are elevated in ocular rosacea, whereas TNF-alpha is not present in either case. The reduced tear turnover, LN(TCR), its inverse correlation with IL-1alpha, and the absence of TNF-alpha in the tears of these patients suggest that the increased concentration of IL-1alpha observed may be largely because of clearance failure of cytokine normally produced at the ocular surface.

Alterations of Ocular Surface Gene Expression in Sjögren’s Syndrome

We have demonstrated that conjunctival epithelium of SS patients displays increased numbers of S-phase cells compared with non-dry eye controls. Moreover, in SS patients, these S-phase cells are distributed throughout all strata of the epithelium. The expression of MUC-1, a cell surface marker indicative of terminally differentiated epithelium, is localized to the conjunctival epithelial surface in SS and control patients. However, MUC-1 surface immunoreactivity appears to be reduced in SS epithelium, suggesting disruption of normal epithelial differentiation. A MUC-1 epitope exposed by pretreatment with neuraminidase is expressed in the basal and suprabasal layers of both patient populations. This antigen likely represents nascent, partially processed MUC-1(6) and may serve as a marker of the preterminally differentiated epithelial phenotype. Messenger RNA encoding several different inflammatory cytokines, including TNF-alpha, IL-1 alpha and beta, IL-6, IL-8, IL-10, and TGF-beta 1, is expressed at elevated levels within the conjunctival epithelium of SS patients compared with non-dry eye controls. Based on these observations, we have formulated a model to explain the ocular surface pathology of Sjögrens syndrome. We hypothesize that mechanical abrasion secondary to aqueous tear deficiency creates an inflammatory environment where conjunctival epithelial cells and lymphocytes are stimulated to produce and secrete various cytokines (i.e., IL-1, TNF-alpha, IL-6, IL-8, etc.) into the tear film. Elevated cytokine levels within the tear film, perhaps combined with reduced concentrations of essential lacrimal gland-derived factors (i.e., EGF, retinol), create an environment in which terminal differentiation of the ocular surface epithelium is impaired. As a consequence, the epithelium becomes hyperplastic, displaying increased mitotic activity, and loses the ability to express mature protective surface molecules including the membrane-bound mucin, MUC-1. This would imply that anti-inflammatory medications (i.e., corticosteroids or cyclosporine) that suppress the inflammatory component of this cascade may ameliorate the ocular surface disease and discomfort experienced by SS patients.

TRANSFORMING GROWTH FACTOR BETA-1 AND BETA-2 IN HUMAN TEAR FLUID

PURPOSE To evaluate human tear fluid for transforming growth factor beta isoforms 1 and 2 (TGF-beta1 and TGF-beta2). METHODS To accomplish this, human tears were evaluated for TGF-betas by quantitative antibody sandwich ELISA (sELISA), mink lung epithelial cell (MLEC) growth inhibition bioassay and western blotting. Various physical and chemical treatments were used to activate TGF-beta in these assays. RESULTS TGF-betas could not be detected in untreated or heated tears by sELISA; however, mean TGF-beta1 concentrations of 2.32 ng/ml were detected in acid-activated tears by sELISA. Furthermore, 10.54 ng/ml of TGF-beta1 and 2.98 ng/ml of TGF-beta2 were detected in tears treated with the mucolytic agent, acetylcysteine. Total TGF-beta bioactivity in human tears measured by the MLEC assay was found to be 13.04 ng/ml in untreated tears and 24.85 ng/ml in acid-activated tears. Approximately one-half TGF-beta in tear specimens was biologically active (mean = 52%, range 39-71%). Total tear TGF-beta bioactivity could be completely neutralized by recombinant human TGF-beta1 latency associated peptide (rh TGF-beta1 LAP). Mean neutralization of tear TF-beta bioactivity was 83% by TGF-beta1-specific antisera, and was 13% by TBF-beta2-specific antisera. Immunoreactive TBF-beta bands at approximately 12.5 and 95 kD were observed in immunoblots of reduced acidified tears. A high molecular weight (MW) TGF-beta band (>203 dD) was noted in untreated tears; however, this band disappeared following treatment with acetylcysteine. CONCLUSIONS The results of these studies indicate that TGF-beta1 and TGF-beta2 are present in human tear fluid, and TGF-beta1 is the predominant isoform. There appear to be factors in human tears capable of binding TGF-beta.

Production and secretion of transforming growth factor beta (TGF-β) by the human lacrimal gland

PURPOSE Transforming growth factor beta (TGF-beta) isoforms 1 and 2 have recently been detected in stimulated human tear fluid. The purpose of this study was to determine if these TGF-sbeta are produced and secreted by the lacrimal gland. METHODS To accomplish this, reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect expression of TGF-beta1 and TGF-beta2 mRNAs in normal human and rabbit lacrimal gland biopsies. Northern blot analyses were used for comparing the relative levels of expression of these TGF-beta mRNAs in rabbit lacrimal glands. Human lacrimal gland biopsies were evaluated by immunohistochemistry for production of TGF-beta1, TGF-beta1 latency associated peptide (LAP), and TGF-beta2 proteins. Supernatants of unstimulated and carbachol-stimulated human lacrimal gland explant cultures were evaluated for secretion of TGF-beta1 and TGF-beta2 by ELISA: RESULTS TGF-beta1 and TGF-beta2 mRNA expression was found in all human and rabbit lacrimal gland specimens by RT-PCR. A greater level of expression of TGF-beta1 than TGF-beta2 mRNA in the rabbit lacrimal gland was noted by Northern blot. In human lacrimal gland biopsies, TGF-beta1 and TGF-beta1 LAP were detected in acinar and ductal epithelia by immunohistochemistry. TGF-beta2 specific antibodies stained a small percentage of acinar and ductal epithelia, as well as material within the lumens of tubulo-acinar complexes in one-third of these glands. TGF-beta1 was detected in supernatants of human lacrimal gland explants, and the concentration of TGF-beta1 increased by an average of 280% after carbachol-stimulation (p = 0.004). TGF-beta2 could not be detected in unstimulated or stimulated human lacrimal gland supernatants. CONCLUSIONS The results of these experiments indicate that TGF-beta1 and TGF-beta2 are produced by and TGF-beta1 is secreted by the human lacrimal gland. They also suggest that the lacrimal gland may be one source of TGF-beta in human tear fluid.

A NOVEL METHOD OF TEAR COLLECTION : COMPARISON OF GLASS CAPILLARY MICROPIPETTES WITH POROUS POLYESTER RODS

PURPOSE To develop a rapid, user-friendly method of tear collection to facilitate tear-protein analysis. METHODS Tears were collected from a total of 19 normal volunteers without evidence of ocular-surface disease with either porous polyester rods or glass-capillary micropipettes. Tear-collection rate and recovery of two tear proteins, epidermal growth factor (EGF, low abundance) and lactoferrin (LFR, high abundance) were compared between polyester rods and glass-capillary micropipettes. The recovery of LFR and EGF and the stability of these proteins after storage at -70 degrees C were quantitated by specific monoclonal enzyme-linked immunosorbent assay (ELISA). RESULTS Polyester rods collected tears an average of 3.9-fold faster than glass-capillary micropipettes (p < 0.001). Both methods were comparable in efficacy of protein recovery. The polyester rods demonstrated a trend toward enhanced recovery, but this difference was not statistically significant (p = 0.12, LFR; p = 0.055, EGF). Analysis of the reliability and reproducibility of the tear-collection assay system revealed that ELISA analysis is highly reproducible, but there is significant day-to-day variation in tear-protein levels of both LFR and EGF for a given volunteer. Both LFR and EGF displayed a trend toward enhanced detection by ELISA shortly after freezing at -70 degrees C and slow decay after storage at -70 degrees C for up to 72 and 105 days, respectively. After stimulation of reflex tearing via the nasolacrimal reflex, LFR levels remained relatively constant, whereas EGF levels for most patients declined and then plateaued. CONCLUSIONS Polyester rods provide a more rapid, user-friendly alternative to glass-capillary micropipettes for the collection and analysis of tear fluid and tear proteins. Polyester rods may have greater clinical utility, facilitating routine analysis of the preocular tear film.

Cytokines and Tear Function in Ocular Surface Disease

In summary, tear EGF levels correlate most strongly with tear production in normals, and it is likely that some form of homeostatic mechanism exists to provide a constant supply to the ocular surface. Commercial ELISA kits appear to measure EGF in tears with good consistency and may be useful in the future to improve comparability of data from different studies. In addition, in ocular rosacea, which mimics keratoconjunctivitis sicca in a number of respects, there is a differential increase in the level of the inflammatory cytokine IL-1 alpha in the tear fluid. Much of this elevation appears to be the result of reduced tear turnover, which may form an important positive feedback mechanism encouraging tear stagnation and the perpetuation of ocular surface inflammation.

THE EFFECTS OF AGE, GENDER, AND FLUID DYNAMICS ON THE CONCENTRATION OF TEAR FILM EPIDERMAL GROWTH FACTOR

PURPOSE To identify the relationship between epidermal growth factor (EGF) concentration in human tears and clinical tear-flow parameters and how these vary with age and gender. METHODS Tear samples were collected with minimal stimulation from 68 healthy and asymptomatic adults (33 men, 35 women), aged 21-88 years. EGF concentrations were determined by sandwich enzyme-linked immunosorbent assay (ELISA) in 65 cases. Schirmer tests were performed without anesthesia, and the clearance of fluorescein from the tear film assessed. The Tear Function Index (TFI) was calculated from these values. RESULTS There were approximately equal numbers of male and female subjects with a similar age distribution for each gender (48 +/- 3 and 51 +/- 3 years, mean +/- SEM, respectively). Ninety percent of tear EGF concentrations were between 0.75 and 7.1 ng/ml. Tear EGF level correlated significantly with Schirmer I value, but not with age. Schirmer I value correlated with tear clearance [LN(TCR)] but not with age. Tear EGF concentrations were significantly higher for men (3.4 +/- 0.3 ng/ml) than for women (2.4 +/- 0.3 ng/ml; p = 0.043). CONCLUSIONS EGF concentrations is tear samples from normal humans were found to correlate with gender and Schirmer I value but not with tear clearance.

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.