The differences of tear function and ocular surface findings in patients with atopic keratoconjunctivitis and vernal keratoconjunctivitis

BACKGROUND: The pathogenesis of the ocular surface disease in atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) has not been fully understood. We tried to clarify the differences in the ocular surface status in patients with AKC, VKC, and healthy control subjects. METHODS: Twenty-four eyes of 12 AKC patients, 12 eyes of six VKC patients, and 20 eyes of 10 normal control subjects were studied. The subjects underwent corneal sensitivity measurements, Schirmer test, tear film break-up time (BUT), vital staining of the ocular surface, conjunctival impression and brush cytology. Impression cytology samples underwent periodic acid Schiff staining for goblet cell density, squamous metaplasia grading, and immunohistochemical staining for MUC1, 2, 4, and 5AC. Brush cytology specimens underwent staining for inflammatory cell counting and Real Time PCR for MUC1, 2, 4, and 5AC mRNA expression. RESULTS: The mean BUT, corneal sensitivity, and conjunctival goblet cell density values in AKC patients were significantly lower compared with VKC patients and control subjects. The squamous metaplasia grades in eyes with AKC were significantly higher compared to eyes with VKC and controls. The inflammatory cell response in brush cytology specimens was different between patients with AKC and VKC. Eyes with AKC showed significantly higher MUC1, 2 and 4 and lower MUC5AC mRNA expression compared to eyes with VKC. CONCLUSIONS: Differences of the infiltrates, higher level of tear instability, lower corneal sensitivity, up-regulation of MUC1, 2, and 4, and down regulation of MUC5AC were important differential features of the ocular surface disease in AKC compared with VKC.     

Clinical grading of vernal keratoconjunctivitis

PURPOSE OF REVIEW: The purpose of the present review is to provide an overview on the clinical features of vernal keratoconjunctivitis on the basis of cases series presented in the literature. Furthermore, a new grading system of vernal keratoconjunctivitis based on the severity of the disease is proposed. Different treatment options are discussed based on the clinical grade of vernal keratoconjunctivitis. RECENT FINDINGS: Recent epidemiological studies on the demographic, clinical and immunologic features of vernal keratoconjunctivitis are presented. The efficacy and complications of treatments are described. SUMMARY: Diagnosis and treatment of patients is a challenge for ophthalmologists as no precise diagnostic criteria have been established, the pathogenesis is unclear, and antiallergic treatments are often unsuccessful. This review describes old and new concepts of vernal keratoconjunctivitis diagnosis and treatment: the clinical features, the diagnostic criteria, the common features between this and other ocular allergies and the therapeutic strategies. On the basis of this knowledge, a new grading system is introduced based on clinical signs and symptoms of ocular surface inflammation. This new grading of vernal keratoconjunctivitis may help clinicians and researchers to classify disease activity and to establish a common agreement for treatments.     

A flow cytometric study about the immunopathology of vernal keratoconjunctivitis

Background: Vernal keratoconjunctivitis (VKC) is a bilateral seasonal conjunctival inflammation. Exact pathogenesis of the disease is unknown, but some evidences suggest T_H lymphocyte–mediated immune reactions. Objective: The aim of this study was to investigate the role of the T lymphocyte and its subsets in the pathogenesis of VKC. Methods: We obtained tear samples from patients with VKC and normal volunteers during active (spring) and quiescent (winter) periods. The patients' records were also obtained for assessment of symptom scores. The percentages of CD4/29+, CD4/45RA+, CD4+, and CD8+ in tear samples were established by using flow cytometry, and the results of all three groups were compared with each other by using the Wilcoxon matched-pair signed-rank test and the Mann-Whitney U test. Results: The percentages of CD4/29+ and CD4+ cells in tears of patients with VKC increased significantly in the active period and decreased to normal levels in the quiescent stage. In contrast, the percentages of CD4/45RA+ and CD8+ cells in tears of patients with VKC did not show any significant change between spring and winter. The patients' symptoms were significantly lower in the quiescent period (winter) compared with the active stage (spring). Conclusion: We propose that increased numbers of CD4+ and CD4/29+ cells in tears may be exacerbating the disease during the spring season. (J Allergy Clin Immunol 1998;101:821-824.)     

Efficacy and safety of cyclosporine eyedrops in vernal keratoconjunctivitis.

BACKGROUND: Corticosteroids have been the only effective topically administered treatment for severe vernal keratoconjunctivitis (VKC), but their prolonged use is often associated with complications. Topical cyclosporine therapy has been used in the past decade, but few controlled trials have been conducted, and conflicting results have been reported. OBJECTIVE: This study sought to evaluate the efficacy and safety of ocular administration of cyclosporine in VKC. METHODS: Twenty-four children with severe VKC were treated with cyclosporine 2% eyedrops. The treatment began in spring and lasted 4 months. One eye was treated with cyclosporine (Cs-eye); the fellow eye received the vehicle as placebo (Pl-Cs-eye) during the first 2 weeks in a double-blind, placebo-controlled trial and thereafter was treated with cyclosporine (open trial). Patients were instructed to protect their eyes against sunlight. Ocular symptoms and signs were scored at entry and at 2 weeks, 4 weeks, and 4 months after the beginning of treatment. All children underwent biochemical and immunologic evaluations. RESULTS: Compared with baseline, scores for ocular signs and symptoms at 2 weeks decreased significantly in the Cs-eyes (P < 0.001), and signs improved in the Pl-Cs-eyes (P = 0.001). A significant difference was noted between Cs-eyes and Pl-Cs-eyes at 2 weeks for both subjective (P < 0.005) and objective (P < 0.001) scores. At 4 weeks, scores for signs (P < 0.001) and symptoms (P = 0.01) were reduced in the Pl-Cs-eyes, with no further improvement in the Cs-eyes. At 4 months, clinical scores had declined further, and serum eosinophil cationic protein levels were significantly lower than at entry (P = 0.009). Most patients reported mild burning sensation and tearing after administration of cyclosporine. Four patients (17%) required an additional brief period of topical corticosteroid therapy. CONCLUSIONS: Cyclosporine eyedrops were effective and safe for treating severe VKC, without causing major side effects. Most of the therapeutic effect was achieved after 2 weeks. The initial therapeutic effect was maintained during the next 3 months, with a further slow decrease in the symptoms.     

Cytokines,matrix metalloproteases,angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patients

Background: To detect the presence of multiple mediators and growth factors in tears of vernal keratoconjunctivitis (VKC) patients with active disease using stationary phase antibody arrays. Methods: Tears were collected from 12 normal subjects (CT) and 24 active VKC patients. Tears were centrifuged and successively probed using three microwell plate arrays specific for: (i) cytokines: interleukin (IL)‐2, IL‐4, IL‐5, IL‐8, IL‐10, IL‐12, IL‐13, interferon‐γ and tumour necrosis factor‐α; (ii) growth factors: basic fibroblast growth factor (bFGF), platelet‐derived growth factor, thrombopoietin, angiopoietin‐2, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), keratocyte growth factor, tissue inhibitor of metalloprotease (TIMP)‐1 and heparin‐binding epithelial growth factor (HB‐EGF) and (iii) matrix metalloprotease (MMP)‐1, MMP‐2, MMP‐3, MMP‐8, MMP‐9, MMP‐10, MMP‐13, TIMP‐1 and TIMP‐2. Results: Interleukin‐8 signals were detected in all CT and highly detected in all VKC samples. The Th2‐type cytokines, IL‐4, IL‐5 and IL‐10 were detected only in tears of VKC patients. Signals for bFGF, HB‐EGF, VEGF and HGF were detected in 41–87% of VKC samples and in few CT samples. Only TIMP‐1 and TIMP‐2 were found in all normal and patient tear samples, whereas MMP‐1, MMP‐2, MMP‐3, MMP‐9 and MMP‐10 were highly present in all VKC samples. Conclusions: Stationary phase antibody array methodology was useful for the screening of various cytokines, growth factors and MMPs in tears. These analyses identified in tears of VKC patients previously unreported factors including MMP‐3 and MMP‐10 and multiple proteases, growth factors and cytokines, which may all play an important role in the pathogenesis of conjunctival inflammation.     

Specific immunoglobulin E antibodies in tear secretions of patients with vernal conjunctivitis

Twenty-two patients with vernal conjunctivitis (VC) were studied by radioallergosorbent test (RAST) for specific IgE antibodies to the inhalant pollen allergens in tear secretions. Specific IgE antibodies were detected in the tear secretions of 12 (54.5%) patients with VC. Six patients had a positive tear RAST, but the corresponding serum RAST and immediate skin test reactivity were negative. The specific tear IgE antibodies appeared to correlate with the seasonal prevalence of the pollen in the environment and the period of maximal symptomatology. Six patients had both positive serum and tear RAST determinations. A double ratio formula with transferrin as a marker for the leakage of plasma proteins into tear secretions and the “specific activity” ratios of IgE antibody to total IgE between the tear secretions and the serum indicated that almost all (79% to 99%) of the tear IgE antibodies were locally produced. IgE antibodies were also detected in the tears of four of 10 patients with allergic conjunctivitis (AC) and of three with allergic rhinitis (AR). However, the “specific activity” ratio in the patients with AC and AR with measurable tear IgE suggested that the IgE antibodies were derived from the leakage of serum proteins into the tear secretions. These findings suggest that the pathogenesis of VC may be IgE mediated in some patients and lends support to the concept that specific IgE antibodies can be produced locally by the target organ, i.e., conjunctival tissues.     

Gelatinase activity of matrix metalloproteinases in the cerebrospinal fluid of various patient populations

We have studied the enzymatic gelatinolytic activity of matrix metalloproteinases (MMPs) present in cerebrospinal fluid (CSF) of samples obtained from 67 individuals, twenty-one nonneurological patients (considered controls) and 46 subjects with various neurological disorders e.g., vascular lesions, demyelination, inflammatory, degenerative and prion diseases. Biochemical characterization of MMPs, a family of neutral proteolytic enzymes involved in extracellular matrix modeling, included determination of substrate specificity and Ca+2 dependency, as well as the effects of protease inactivators, carboxylic and His (histidine) residue modifiers, and antibiotics. Whereas all CSF samples expressed MMP-2 (gelatinase A) activity, it corresponded in most cases (normal and pathological samples) to its latent form (proenzyme; pMMP-2). In general, inflammatory neurological diseases (especially meningitis and neurocisticercosis) were associated with the presence of a second enzyme, MMP-9 (or gelatinase B). Whereas MMP-9 was found in the CSF of every tropical spastic paraparesis patient studied, its presence in samples from individuals with vascular lesions was uncommon. Patients blood-brain barrier damage was ascertained by determining total CSF protein content using both, the conventional polyacrylamide gel electrophoresis procedure under denaturing conditions and capillary zone electrophoresis.     

Matrix metalloproteases in vernal keratoconjunctivitis, nasal polyps and allergic asthma

BACKGROUND: Allergic conditions in different organs share many similarities in their inflammatory response. Vernal keratoconjunctivitis (VKC), asthma and nasal polyps exhibit several similar, but site-specific mucosal structural changes. The aim of the study was to investigate whether matrix metalloproteases contribute to different tissue remodelling aspects in different organs. METHODS: Mucosal biopsies were obtained from conjunctiva of healthy donors, tarsal conjunctiva of vernal patients, bronchi of non-asthmatic subjects, bronchi of mild stable asthmatic patients, nasal mucosa of non-allergic donors and nasal polyps of allergic patients. Distribution of metalloprotease-1, -3, -9, -13, tissue inhibitor of metalloproteases-1, collagens I and III and the presence of eosinophils and CD4+ cells were evaluated by immunohistochemistry. RESULTS: Collagens were highly diffuse in the giant papillae of VKC and in nasal polyps, and yet less increased in the subepithelium of asthmatic patients. Immunostaining for metalloprotease-1, -3, -9 and -13 was significantly higher in VKC compared with normal conjunctiva. Metalloprotease-9 staining was higher in the stroma of polyps vs. normal nasal mucosa, and only metalloprotease-13 was significantly more expressed in asthmatic vs. non-asthmatic subjects. Metalloprotease-9 immunostaining was more intense in vernal compared with other tissues. In all pathological tissues, metalloprotease-9-positive staining was in association with eosinophils and CD4+ cells. CONCLUSIONS: Expression of metalloproteases may play an important role in inducing the structural changes seen in VKC, nasal polyps and asthma. Tissue remodelling and gelatinase immunoexpression was more dramatic in giant papillae of vernal patients compared with other tissue sites of chronic allergic inflammation.     

Elevated levels of substance P in tears of patients with allergic conjunctivitis and vernal keratoconjunctivitis

Background Recetit studies have suggested that the nervous system may participate in inflammatory processes. Substance P (SP) acts as a chemical mediator as well as a neurotransmitter. Objective In order to clarify the pathogenesis of ocular allergic diseases, we assessed the concentration of SP in tears. Methods Using a highly sensitive and specific double-antibody enzyme immunoassay (EIA), we determined the SP concentration in tears of 10 patients with seasonal allergic conjunctivitis, 10 with atopic dermatitis without keratoconjunctivitis (AD), 13 with vernal keratoconjunctivitis (VKC) and 65 normal controls. Giemsa's staining for brush cytology samples and histocytological study by immunocytochemical staining of giant papillary conjunctival cells from VKC and normal controls was conducted. Results The mean SP level was low in the normal controls and AD, whereas patients with seasonal allergic conjunctivitis and VKC showed significant elevation of SP (P<0.01). Brush cytology samples showed conjunctival epithelial cells with lymphocytes, neutrophils and eosinophils that were not seen in normal subjects. Histocytological examination demonstrated SP positive cells in the conjunctiva of patients with VKC, but not in normal controls. Conclusion This study suggests that the increased level of SP in tears may contribute to the pathogenesis and severity of ocular allergic diseases.     

Measurement of IL-4 in tears of patients with seasonal allergic conjunctivitis and vernal keratoconjunctivitis.

To elucidate the mechanism of ocular surface allergic disease, we focused on IL-4, which is one of the key factors in regulating IgE production, and thus determined the concentration of IL-4 in tears. IL-4 concentration was determined in the tears of 15 patients with seasonal allergic conjunctivitis, 15 vernal keratoconjunctivitis (VKC), 10 giant papillary conjunctivitis (GPC), 10 patients with non-allergic conjunctivitis and post-cataract surgical conjunctivitis as intermediate conjunctivitis, and 10 normal subjects using a highly sensitive sandwich ELISA. The mean level of IL-4 in normal controls was low, and seasonal allergic conjunctivitis, VKC and GPC showed a significant elevation (P < 0.05), respectively. IL-4 of VKC and GPC were also significantly higher than allergic conjunctivitis, and non-allergic conjunctivitis and post-cataract surgical conjunctivitis were not higher than normal. These results raise the possibility that the increased level of IL-4 in tears could play a role in allergic disease and its severity in patients.     

Expression of matrix metalloproteinases and their tissue inhibitors in the serum and cerebrospinal fluid of patients with meningitis

Meningitis is associated with an imbalance between matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of MMP (TIMPs). Serum and CSF were collected prospectively from all patients with meningitis between January 2008 and December 2008 to measure the concentrations of MMP/TIMP in those patients who underwent a lumbar puncture for a presumptive diagnosis of meningitis. A total of 199 patients were enrolled into the study. The concentrations of CSF MMP‐9 and TIMP‐1 were significantly higher in the meningitis group compared with the control group (p 0.032 and p <0.001, respectively). However, the CSF TIMP‐4 levels were significantly lower in the meningitis groups compared with the control groups (p <0.001). Patients with bacterial meningitis had higher CSF MMP‐9 and TIMP‐1 levels than those who had aseptic meningitis and controls. Patients with various infectious meningitis etiologies tended to have higher CSF MMP‐9 expression by gelatin zymography when compared with the controls. In conclusion, MMP/TIMP system dysregulation was found in patients with meningitis, and CSF MMP and TIMP might act as novel indicators in patients with meningitis.     

Eosinophil cationic protein in tears of normal subjects and patients affected by vernal keratoconjunctivitis

The objectives of this study were to determine: 1) levels of tear eosinophil cationic protein (ECP) in patients with vernal keratoconjunctivitis (VKC); 2) the effect of pharmacologic therapy on ECP release; and 3) the correlation of this mediator with the severity of the disease. Tears were collected from 10 controls and 20 VKC patients before and after therapy for cytologic analysis and ECP measurement by radioimmunoassay. Ocular signs and symptoms were evaluated before tear collection. Mean ECP levels in controls were 7.5 ± 0.4 μg/l, and in VKC patients, 988.3 ± 128 μg/l before therapy ( P <0.001) and 566.3 ± 121 μg/l after therapy ( P <0.005). In dexamethasone (Dex) 0.1%, or cyclosporin A (CsA) 2%, patients (five per group), tear ECP decreased significantly after 7–14 days of treatment. Disodium cromoglycate (DSCG) 4% (five patients) for 14 days did not significantly affect ECP levels. ECP levels were significantly correlated with allergic signs ( P <0.001), symptoms ( P <0.001), and the number of eosinophils in tears (P<0.005). The results of this study suggest that tear ECP levels accurately reflect the clinical status of VKC patients. The measurement of ECP may prove useful not only in the diagnosis and monitoring of allergic disease, but also as an objective parameter for the evaluation of new antiallergic therapies.     

Direct fluorescent antibody assay and polymerase chain reaction for the detection of Chlamydia trachomatis in patients with vernal keratoconjunctivitis

OBJECTIVES:

To identify Chlamydia trachomatis via polymerase chain reaction and a direct fluorescent antibody assay in patients with vernal keratoconjunctivitis while comparing the efficacies of both tests for detecting Chlamydia trachomatis in these conditions.

METHODS:

Conjunctival scraping samples were obtained from 177 patients who were divided into two groups: a vernal keratoconjunctivitis group (group A) and a control group (group B). The polymerase chain reaction and a direct fluorescent antibody assay were performed. Sensitivity, specificity, receiver operating characteristic curves, and areas under the curve were calculated for both tests in groups A and B. Receiver operating characteristic curves were plotted using a categorical variable with only two possible outcomes (positive and negative).

RESULTS:

Statistical analysis revealed a significant association between vernal keratoconjunctivitis and Chlamydia trachomatis infection detected by a direct fluorescent antibody assay with high sensitivity and specificity. All patients in group A with positive polymerase chain reactions also presented with positive direct fluorescent antibody assays.

CONCLUSION:

The association between vernal keratoconjunctivitis and Chlamydia trachomatis infection was confirmed by positive direct fluorescent antibody assays in 49.4% of vernal keratoconjunctivitis patients and by positive polymerase chain reactions in 20% of these patients. The direct fluorescent antibody assay detected Chlamydia trachomatis in a higher number of patients than did the polymerase chain reaction. Although the diagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients. Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggest considering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergic disease.     

Extracellular matrix remodelling: the role of matrix metalloproteinases

Matrix metalloproteinases (MMPs) are a growing family of metalloendopeptidases that cleave the protein components of the extracellular matrix and thereby play a central role in tissue remodelling. For many years following their discovery, MMPs were believed to function primarily as regulators of ECM composition and to facilitate cell migration simply by removing barriers such as collagen. It is becoming increasingly clear, however, that MMPs are implicated in the functional regulation of a host of non-ECM molecules that include growth factors and their receptors, cytokines and chemokines, adhesion receptors and cell surface proteoglycans, and a variety of enzymes. MMPs therefore play an important role in the control of cellular interactions with and response to their environment in conditions that promote tissue turnover, be they physiological, such as normal development, or pathological, such as inflammation and cancer. This review summarizes some of the recent discoveries that have shed new light on the role of MMPs in physiology and disease.