Reduced structural proteins in the conjunctival epithelium in allergic eye disease.

AIMS: Allergic eye disease affects up to 20% of the population with varying severity. The conjunctival epithelium plays a key role in allergic eye disease. The purpose of this study was to determine whether the conjunctival epithelium is abnormal in allergic eye disease. METHODS: Conjunctival biopsy samples were taken from patients with seasonal allergic conjunctivitis (SAC) 'in' and 'out of season' and nonatopic control subjects. Specimens were fixed in glycol methacrylate, 2 microm serial sections cut and Image-J used to assess the sites and areas of immuno-staining. RESULTS: E-cadherin, CD44, keratins K5/6, K8, K13, K14, K18 and pan-keratin immuno-staining were all significantly lower in patients 'out of season' compared with normal controls. No structural differences in the epithelium were observed between the two groups. The epithelium of patients 'in season' was thicker and immuno-staining of the above markers similar to controls. CONCLUSIONS: The expression of a wide spectrum of epithelial cell adhesion proteins and cytoskeletal elements is downregulated in the conjunctiva of SAC patients 'out of season' compared with normal controls. We suggest that this could have an important impact on the ability of the epithelium to protect itself against allergen penetration, potentially influencing the development and course of allergic eye disease and offering a novel area for therapeutic control.

A randomised, double-blind trial of topical ketorolac vs artificial tears for the treatment of episcleritis.

PURPOSE: To determine whether topical ketorolac (Acular) is more effective than artificial tears in treating the signs and symptoms of idiopathic episcleritis. METHODS: In this prospective, randomised, double-blind study, 38 eyes of 37 patients presenting with idiopathic episcleritis were allocated to receive either topical ketorolac (0.5%) or artificial tears three times a day for 3 weeks. The severity of patients' signs (episcleral injection and the number of clock hours affected) were recorded at weekly intervals. Patients' symptoms (perceived redness and pain scores) were recorded using a daily diary. RESULTS: There was no significant difference in the ophthalmic signs between the two groups at each assessment, including intensity of episcleral injection and the number of clock hours affected. No significant difference was found in the time to halve the baseline redness intensity scores (4.4 vs 6.1 days, P=0.2) or pain scores (3.6 vs 4.3 days, P=0.55). Significantly more patients on ketorolac reported stinging at the first follow-up visit (P<0.001). CONCLUSION: Topical ketorolac is not significantly better than artificial tears in treating the signs or symptoms of idiopathic episcleritis.

Nedocromil sodium and levocabastine reduce the symptoms of conjunctival allergen challenge by different mechanisms.

BACKGROUND: Nedocromil sodium and levocabastine are widely used for the treatment of ocular allergy, but their mechanisms of action are unclear. OBJECTIVE: We sought to compare the efficacy and mechanisms of action of nedocromil sodium and levocabastine in reducing conjunctival symptoms after ocular allergen challenge. METHODS: We performed a double-blind, placebo-controlled study in which 48 subjects were randomized to 3 groups to receive nedocromil sodium (2%), levocabastine (0.05%), or placebo eye drops twice daily for 2 weeks before ocular challenge with 10 microL of ryegrass extract. Symptoms and tear histamine and PGD(2) concentrations were determined before challenge and at 10, 20, 30, 60, 180, and 360 minutes after challenge. Bulbar biopsy specimens were taken at 6 and 24 hours after challenge to assess conjunctival inflammatory cell numbers, adhesion molecule expression, and mast cell-associated IL-4, IL-5, IL-6, IL-13, and TNF-alpha levels. RESULTS: Both drugs significantly reduced total symptom scores (P <.05) at all times after challenge compared with placebo. Itching, hyperemia, and lacrimation were most affected. Nedocromil sodium treatment reduced tear concentrations of histamine (by 77%) and PGD(2) (by 70%) at 30 minutes after challenge (both P <.05). In biopsy specimens nedocromil sodium reduced the number of 3H4-positive mast cells (purportedly the secreted form of IL-4) by 49% at 6 hours and 59% at 24 hours (both P <.05). Levocabastine reduced intercellular adhesion molecule 1 expression by 52% at 6 hours and 45% at 24 hours (both P <.05). CONCLUSION: Nedocromil sodium and levocabastine both reduced the conjunctival symptoms after ocular allergen challenge but appeared to work by different mechanisms. Nedocromil sodium reduced mast cell function, whereas levocabastine appeared to have primarily antihistaminic actions, although it also reduced the expression of intercellular adhesion molecule 1.

The relative contribution of mast cell subsets to conjunctival TH2-like cytokines.

PURPOSE: To investigate the distribution of the T-helper (TH)2-like cytokines, interleukin (IL)-4, IL-5, IL-6, and IL-13 between mast cell subsets in conjunctival biopsy specimens from normal subjects and those with seasonal allergic conjunctivitis (SAC) during and outside of the grass pollen season. METHODS: Sequential and double in situ hybridization (ISH) and immunohistochemistry (IHC) were performed on thin sections of human conjunctiva to determine the colocalization of the immunoreactivity of IL-4, IL-5, IL-6, and IL-13 to mast cell subsets in normal subjects and subjects with atopy and to detect IL-4 mRNA in conjunctival mast cells. RESULTS: More than 90% of IL-4+-immunoreactive cells were observed to be mast cells in conjunctival biopsy specimens from all patient groups. The majority of IL-5+, IL-6+, and IL-13+ cells were also noted to be mast cells for each group. IL-4 preferentially colocalized to the tryptase+-chymase+ mast cell phenotype (MC(TC)) with MC(TC) cells comprising 93.3% of cytokine+ mast cells in symptomatic SAC (P = 0.0017), 89.2% in asymptomatic SAC (P = 0.0008), and 77.8% in normal subjects (P = 0.0472). IL-13 appeared to colocalize preferentially to the MC(TC) phenotype and IL-5 and IL-6 to the MC(T) phenotype. ISH showed that 75.8% of mast cells in normal subjects, 78.7% in subjects with symptomatic SAC, and 18.7% in subjects with asymptomatic SAC expressed mRNA for IL-4. CONCLUSIONS: Conjunctival mast cells are an important source of IL-4, IL-5, IL-6, and IL-13 immunoreactivity, with preferential colocalization of IL-4 and IL-13 on the MC(TC) subset and IL-5 and IL-6 to the MC(T) subset. This evidence suggests that differences in protease phenotype may also reflect functional differences evidenced by the different patterns of cytokine distribution.

Nedocromil sodium inhibits histamine-induced itch and flare in human skin.

This study was designed to test the hypothesis that nedocromil sodium inhibits sensory nerve function to reduce flare and itch in human skin. Nedocromil sodium (2%) or water (control) was introduced into the volar forearm skin of eight non-atopic volunteers by iontophoresis (8 mC) and histamine (20 microl of 1 microM and 300 nM) injected intradermally 10 min later at the same site. Itch was assessed on a visual analogue scale every 20 s for 5 min. Weal and flare areas and mean blood flux within the flare were assessed by scanning laser Doppler imaging at 10 min. The results showed that nedocromil sodium reduced itch scores, totalled over 5 min, by approximately 74.0% (P<0.005) and flare areas by approximately 65% (P<0.03). Neither weal areas nor blood flux within were reduced. These data demonstrate that nedocromil sodium is effective in reducing neurogenic itch and flare in the skin. We suggest that its mechanism of action is modulation of sensory neurone activation or conduction in the skin.

Tear and conjunctival changes during the allergen-induced early- and late-phase responses.

BACKGROUND: Allergic eye disease is common, but little is known about the underlying disease mechanisms. Conjunctival allergen challenge causes symptoms similar to those of seasonal allergic conjunctivitis and is a useful model to study. OBJECTIVE: We have used allergen challenge to investigate the course of the ocular response, tear inflammatory mediators, tissue adhesion protein expression, and cellular infiltration. METHODS: Eighteen atopic patients and 4 nonatopic control subjects were challenged with extracted mixed grass or Dermatophagoides pteronyssinus in one eye and control vehicle in the other. The clinical response was recorded, and tears were collected over a 6-hour period. Conjunctival biopsy specimens were taken from the challenged eye at 6 or 24 hours. RESULTS: An early-phase response (maximal at 20 minutes) showed a significant increase in tear histamine and tryptase levels, reducing to control levels again by 40 minutes. At 6 hours, a late-phase response occurred with increased symptoms, a second peak of tear histamine and eosinophil cationic protein but not tryptase, upregulation of the adhesion molecules E-selectin and intercellular adhesion molecule, and a cellular infiltrate of mast cells, neutrophils, eosinophils, macrophages, and basophils, with T cells increased only in bulbar biopsy specimens. CONCLUSIONS: The early peaks of tear histamine plus tryptase indicate that the mast cell is responsible for the early-phase response, but basophils may be involved in the late-phase response. Both tear and biopsy findings underline the significance of the late-phase response as the transition between a type I response and clinical disease.

Polymerase chain reaction analysis of corneal epithelial and tear samples in the diagnosis of Acanthamoeba keratitis.

PURPOSE: Acanthamoeba is an uncommon cause of corneal infection in which the best visual outcome follows prompt diagnosis and a long course of appropriate antimicrobial therapy. Because conventional detection techniques for Acanthamoeba have certain limitations, we investigated the ability of the polymerase chain reaction (PCR) to confirm the clinical diagnosis of Acanthamoeba keratitis, with the ultimate aim of achieving early diagnosis. METHODS: Using two different pairs of primers, PCR was performed on representative cultured Acanthamoeba isolates to confirm the assay's ability to amplify Acanthamoeba DNA from a wide range of acanthamoebae. Subsequently, corneal epithelial samples from 19 patients and tear samples from 12 patients with Acanthamoeba keratitis were analyzed by PCR for the presence of Acanthamoeba DNA. RESULTS: Acanthamoeba DNA was amplified by PCR from 16 (84%) of 19 corneal epithelial samples, whereas Acanthamoeba was cultured from 10 samples (53%), all of which were PCR positive. Tear samples from 8 (66%) of 12 patients were positive on PCR testing, and one tear sample was PCR positive, whereas the corresponding epithelial biopsy had yielded a negative PCR result. Samples from culture-positive patients were positive on PCR testing more frequently than those from culture-negative patients (10/10 culture-positive corneal epithelial and 5/7 [71%] culture-positive initial tear samples versus 6/9 [66%] culture-negative corneal epithelial and 2/5 [40%] culture-negative tear samples). All control epithelial (n = 15) and tear (n = 15) samples yielded negative results. CONCLUSIONS: PCR was a more sensitive diagnostic test than a culture for Acanthamoeba keratitis, and the use of two different primers achieved better sensitivity than a single set. A PCR of a tear sample also may be a useful complementary test and, in combination with PCR of epithelial samples, would prove particularly helpful in confirming the clinical diagnosis in culture-negative cases.

Adhesion molecules and relationship to leukocyte levels in allergic eye disease.

PURPOSE: To evaluate the conjunctival expression of leukocyte cell adhesion molecules (CAMs) and their relationship to leukocyte patterns on the microvasculature in the different clinical subtypes of allergic eye disease. METHODS: Immunohistochemical analysis, using appropriate monoclonal antibodies, was applied to glycolmethacrylate-embedded biopsies of bulbar and tarsal conjunctival tissue. The proportion of total blood vessels expressing a particular CAM was derived and related to individual cell types identified by cell-specific markers, such as mast cells, eosinophils, neutrophils, T cells, and macrophages. Statistical analysis was used to correlate adhesion molecule expression and, ultimately, cell type. RESULTS: There was a basal expression of intercellular adhesion molecule-1 (ICAM-1) (21% bulbar, 18% tarsal), E-selectin (15% bulbar, 21% tarsal), and vascular cell adhesion molecule-1 (VCAM-1) (13% bulbar and tarsal) in normal controls. In seasonal and perennial (bulbar and tarsal conjunctival) allergic tissue, ICAM-1 and E-selectin were expressed in 40% to 78% of vessels; in chronic disease, they were expressed in 45% to 80% of vessels; and in vernal giant papillae, they were expressed in as many as 90% of vessels. There was also increased expression of endothelial VCAM-1 in all forms of allergic eye disease; the greatest values were found in vernal giant papillae (64%). Biopsies taken in winter from seasonal sufferers demonstrated a marked reduction in levels of all three CAMs compared with those taken in the pollen season. This is almost consistent with values found in normal conjunctiva. Positive correlations were found between the levels of ICAM-1 and E-selectin expression and the degree of granulocyte and lymphocyte infiltration, although VCAM-1 expression correlated most closely with eosinophil numbers. CONCLUSIONS: Increased levels of cell adhesion molecules on the microvasculature and the factors that regulate them are likely to be responsible for the infiltration of cells bearing their ligands and may perpetuate inflammation in the chronic forms of allergic eye disease.

Human mast cells express stem cell factor.

Stem cell factor (SCF) is a major cytokine regulator of mast cell growth and function. The present study demonstrates that human mast cells are able to produce SCF. Constitutive synthesis of SCF mRNA was seen in the mast cells isolated from human lung and skin by RT-PCR. This was confirmed by in situ hybridization in conjunctival mast cells of both tryptase-only (MCT) and tryptase/chymase (MCTC) subsets. SCF protein product was found in conjunctival MCT and MCTC mast cells by immunohistochemistry. Soluble SCF protein was detected in the culture supernatant of isolated lung mast cells by ELISA, and cross-linkage of IgE receptor (Fc epsilon-RI) on the lung mast cells in culture did not alter SCF mRNA expression, or the secreted soluble SCF protein. This was consistent with the finding that levels of SCF mRNA expression in conjunctival mast cells were similar between normal subjects and patients with seasonal allergic conjunctivitis (SAC). This study shows that human mast cells themselves are a cellular source of SCF, as well as being target cells for this growth factor. SCF may regulate mast cell growth and function via both paracrine and autocrine mechanisms. The production of SCF by mast cells may be regulated via mechanisms other than IgE receptor-mediated pathways.

Association between nonadministration of subconjunctival cefuroxime and postoperative endophthalmitis.

PURPOSE: To determine whether not administering subconjunctival cefuroxime during cataract surgery is associated with postoperative endophthalmitis. SETTING: Southampton Eye Unit, Southampton General Hospital, England. METHODS: This retrospective, case-control study comprised nine patients who developed endophthalmitis after cataract surgery in a single ophthalmic unit over a 21 month period. Ten control patients for each case were randomly chosen from patients having cataract surgery within 1 week of the endophthalmitis case. RESULTS: None of the nine endophthalmitis patients received peroperative subconjunctival cefuroxime compared with 43 of 90 control patients (47.8%) (P = 0.008). No other variables were found to be associated with development of endophthalmitis in this study. CONCLUSION: Nonadministration of subconjunctival cefuroxime was associated with subsequent endophthalmitis. A further study to determine whether the observed association is causal is therefore warranted.

Seasonal allergic conjunctivitis is accompanied by increased mast cell numbers in the absence of leucocyte infiltration.

BACKGROUND: Seasonal allergic conjunctivitis (SAC) is the most common allergic disease to affect the eye, occurring alone or in association with allergic rhinitis. Infiltration with mast cells and eosinophils is characteristic of the chronic forms of allergic conjunctivitis such as vernal and atopic keratoconjunctivitis, and these cell types also contribute significantly to allergic inflammation in the skin. Indirect evidence for a similar pattern of cellular events in SAC comes from studies which demonstrate raised eosinophil and neutrophil numbers in conjunctival scrapings and elevated levels of mast cell tryptase in tears following allergen challenge. OBJECTIVE: To directly characterize the inflammatory cell infiltrate in SAC and to determine its clinical relevance. METHODS: We employed specific immunohistochemical staining to count mast cells, eosinophils and neutrophils in the conjunctival epithelium and lamina propria of eight atopic patients with SAC in, and 12 SAC patients out of the hay fever season. Sixteen patients with no history of ocular allergy were used as control subjects. RESULTS: Mast cells were absent from normal epithelium. During the pollen season median mast cell numbers in the lamina propria were found to be increased by 61% in patients with SAC compared with normals (P=0.012). Eosinophils were found in the lamina propria in less than half of the symptomatic patients with SAC and in only three patients were eosinophils present in the epithelium. The neutrophil numbers in the lamina propria of patients with SAC tended to be higher than normals but these changes did not reach statistical significance. CONCLUSION: These data based on the direct assessment of conjunctival tissue provide evidence that symptoms occur in SAC in the absence of detectable recruitment of eosinophils or neutrophils. This suggests that this disorder is related to mast cell-mediated changes.

Immunolocalization of cytokines to mast cells in normal and allergic conjunctiva.

BACKGROUND: Recently, the potential role of mast cells in allergic reactions has been extended by the discovery that these cells synthesize, store and secrete multifunctional cytokines. Seasonal allergic conjunctivitis is characterized as an immediate hypersensitivity reaction, in which allergen binds to specific IgE on mast cells, leading to release of pre-formed and newly synthesized inflammatory mediators. OBJECTIVE: In this study we aimed to localize the cytokines IL-4, IL-5, IL-6, IL-8 and TNF alpha to conjunctival mast cells and to examine the relationship between mast cell-associated cytokines and allergic conjunctivitis. METHODS: Immunohistochemistry was performed on serial sections of conjunctival biopsies from patients with seasonal allergic conjunctivitis, in and out of the hay fever season, as well as from non-allergic volunteers. RESULTS: IL-4, IL-5, IL-6 and TNF alpha were localized to mast cells in normal and allergic conjunctiva. IL-8 was localized to mast cells in two patients with seasonal allergic conjunctivitis, one during and the other outside the pollen season. Using the monoclonal antibody 3H4, which identifies the secreted form of IL-4, biopsies from patients with active seasonal allergic conjunctivitis contained a significantly higher proportion of mast cells positive for IL-4, than those from out-of-season patients (P=<0.016). There was no difference between the two groups in the number of mast cells immunostained by the antibody 4D9 which identifies the stored form of IL-4. CONCLUSIONS: These results suggest that conjunctival mast cells can store a range of multifunctional cytokines and release IL-4 during active disease, which may give them an important role in upregulating allergic inflammation in the conjunctiva.

Mast cell distribution and neutral protease expression in acute and chronic allergic conjunctivitis.

Allergic eye disease has a variety of clinical manifestations including seasonal atopic conjunctivitis (SAC), perennial atopic conjunctivitis (PAC), atopic keratoconjunctivitis (AKC), and atopic blepharoconjunctivitis (ABC). We have investigated the number, distribution and protease expression of mast cells in normal and diseased conjunctiva with the use of immunohistochemistry in water-miscible resin sections. The median mast cell densities in normal subjects were 17 mm-2 in the bulbar substantia propria and 9 mm-2 in tarsal substantia propria. Mast cells were absent from the normal conjunctival epithelium at both sites. Mast cell densities were increased in the bulbar substantia propria in SAC, AKC and ABC. Tarsal substantia propria showed a significant increase in mast cells in ABC and AKC disease states. Mast cells express a range of proteases which varies according to their anatomic site. Mast cells in connective tissue are described to contain tryptase, chymase, cathepsin-G and carboxypeptidase-A, whereas mucosal mast cells contain only tryptase. In the diseased conjunctiva there was a marked reduction in proteases other than tryptase in the intraepithelial mast cells. There were also significant reductions in protease expression other than tryptase in the bulbar substantia propria in AKC and ABC. There appear to be specific alterations in the distribution of mast cells in the sub-categories of allergic eye disease. The distinction between mucosal and connective tissue mast cell phenotypes is not clear-cut and may depend on the functional state of the mast cells in relation to the microenvironment.

The origin of keratopathy in chronic allergic eye disease: a histopathological study.

Although the clinical features of atopic keratoconjunctivitis have been well described, it is recognised that some adults with severe chronic allergic conjunctivitis do not develop a keratopathy despite having marked disease of their lid margins and conjunctiva. The aetiology of the keratopathy of chronic allergic eye disease is not known but may result from the release of toxic mediators from mast cells, eosinophils and neutrophils in the inflamed conjunctiva. To examine this theory we have counted these cells in tarsal and bulbar conjunctival biopsies from patients with severe allergic conjunctivitis and compared the numbers in samples from patients with or without keratopathy. Our results indicate that patients with chronic allergic conjunctivitis with keratopathy have higher cell numbers in their conjunctiva than patients with no keratopathy, especially those staining for eosinophil cationic protein in the tarsal epithelium. A statistically significant difference between disease groups could only be demonstrated, however, when the combined totals of mast cells, eosinophils and neutrophils were compared.

Long-term endothelial cell loss and breakdown of the blood-aqueous barrier in cataract surgery.

Progressive endothelial cell loss and endothelial cell loss induced at the time of surgery occurs in all eyes with rigid anterior chamber intraocular lenses (IOLs). Eyes with surgical tuck or late ovaling of the pupil following surgery have greater yearly rates of cell loss than eyes that have no complications. This progressive loss may be related to chronic uveitis from iris chafing by the implant or to direct mechanical damage to the corneal endothelium. We have demonstrated that fluorophotometry shows chronic damage to the blood-aqueous barrier in all eyes with rigid anterior chamber IOLs, but this does not correlate with the degree of endothelial cell loss. Our results suggest there is damage to the blood-aqueous barrier and to the corneal endothelium, but the damage to the latter influences progressive endothelial cell loss.

Mast cell numbers and staining characteristics in the normal and allergic human conjunctiva.

In this study we report the numbers and metachromatic dye-staining characteristics of mast cells (MCs) in the conjunctiva of normal subjects and patients with seasonal allergic conjunctivitis caused by pollenosis. In addition, we have used a monoclonal antibody to the MC-specific enzyme, tryptase, to enumerate tryptase-positive cells immunohistochemically. Tarsal conjunctival MCs were found to be present in increased numbers in the allergic compared to the nonallergic subjects. Most of the MCs exhibited staining that was formaldehyde resistant, compatible with their identification as connective tissue MCs or basophils. The high positive staining for tryptase confirmed that the metachromatic cells stained with toluidine blue were MCs and not basophils, both in normal and allergic subjects.