Andrea Leonardi

Effect of lodoxamide and disodium cromoglycate on tear eosinophil cationic protein in vernal keratoconjunctivitis.

AIM To validate the use of tear eosinophil cationic protein (ECP) as a marker for eosinophil activation, and its pharmacological modulation, in addition to evaluating the efficacy of lodoxamide and sodium cromoglycate in the treatment of vernal keratoconjunctivitis (VKC). METHODS Tears were collected from 30 patients affected by active mild to moderate VKC before and after therapy with disodium cromoglycate 4% (DSCG) (n=15) or lodoxamide 0.1% (n=15) for 10 days. Tear cytology and ECP measurement were performed, and ocular signs and symptoms evaluated. RESULTS While statistically significant changes did not occur after DSCG therapy, mean tear ECP increased from 343 (SD 363) μg/l to 571 (777) μg/l due to marked elevation in six eyes. The clinical score in DSCG eyes did not improve. After lodoxamide therapy, both clinical signs and symptoms, and tear ECP levels (560 (756) μg/l to 241 (376) μg/l) decreased significantly (p<0.0001 and p<0.01, respectively). Compared with DSCG treatment, lodoxamide was more effective in reducing signs and symptoms (p<0.005). ECP levels were significantly correlated with signs, symptoms, corneal involvement, and number of eosinophils in tears (p<0.0001). CONCLUSIONS In patients with VKC, lodoxamide significantly reduced ECP tear levels, and thus, eosinophil activation, and was more effective than DSCG in reducing clinical signs and symptoms.

Cytokine and chemokine levels in tears and in corneal fibroblast cultures before and after excimer laser treatment

PURPOSE: To measure multiple cytokine and chemokine production in tears of myopic patients before and after laser in situ keratomileusis (LASIK) and in human corneal fibroblast (HCF) cultures before and after excimer laser treatment. SETTING: Department of Neuroscience, Ophthalmology Unit, University of Padua, Italy and Vissum‐Instituto de Oftalmológico de Alicante, Alicante, Spain METHODS: Tear samples were obtained from 15 myopic patients before LASIK and 1 and 24 hours after LASIK. Quiescent HCF cultures were treated using the same laser energy. Culture medium was collected before treatment and after 1 and 24 hours. Cytokine concentrations were determined using multiplexed bead analysis. RESULTS: Compared with baseline values, interleukin (IL)‐12 tear levels were significantly increased 1 hour after surgery and eotaxin levels were significantly increased at 24 hours (both P<.05). Culture medium of HCF contained high levels of IL‐6, IL‐8, and monocyte chemotactic protein (MCP)‐1 and low levels of IL‐1, eotaxin, and regulated on activation, normal T expressed, and secreted (RANTES) cytokine. One hour after treatment, levels of all cytokines were significantly reduced. At 24 hours, IL‐1, IL‐6, IL‐8, and MCP‐1 levels were significantly increased compared with values at baseline and at 1 hour while RANTES cytokine and eotaxin levels had returned to baseline levels. CONCLUSIONS: In vivo and in vitro studies showed that after excimer laser treatment, cytokines are released to modulate the wound‐healing process; however, they can potentially induce inflammation. However, these types of in vitro studies, although useful for evaluating changes in cytokine profiles before and after treatment, only partially reproduce in vivo corneal behavior.

Anti-inflammatory and antiallergic effects of ketorolac tromethamine in the conjunctival provocation model

AIM To study the effect of the topical anti-inflammatory drug, ketorolac, on (1) the clinical allergic reaction induced by the conjunctival provocation test (CPT); (2) the release of tryptase in tears; and (3) the expression of adhesion molecules on the conjunctival epithelium. METHODS 10 allergic but non-active patients were challenged in both eyes with increasing doses of specific allergen to obtain a positive bilateral reaction and rechallenged, after 1 week, to confirm the allergic threshold dose response. After 2 weeks, a third CPT was then performed bilaterally 30 minutes after topical application of ketorolac in one eye and placebo in the contralateral eye in a double blind fashion. Clinical symptoms and signs were registered 5, 10, 15, and 20 minutes after challenge. The following objective tests were performed: tear tryptase measurement; tear cytology; and conjunctival impression cytology for immunohistochemical expression of ICAM-1 on epithelial cells. RESULTS Compared with placebo, ketorolac significantly reduced the total clinical score and the itching score in the 20 minutes after challenge (p<0.0005). Tear levels of tryptase were significantly reduced in the ketorolac pretreated eyes compared with placebo (p<0.03). Eosinophils, neutrophils, and lymphocytes in tear cytology were significantly lower in ketorolac treated eyes compared with placebo. A significant difference in the epithelial expression of ICAM-1 was observed between placebo and ketorolac treated eyes (p<0.05). CONCLUSION Ketorolac proved to be effective in reducing mast cell degranulation, as indicated by significantly decreased tryptase tear levels, as well as the clinical and cytological allergic reaction.

Tumor necrosis factor-alpha (TNF-α) in seasonal allergic conjunctivitis and vernal keratoconjunctivitis

Purpose To quantify the presence of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) in allergic conjunctivitis. Materials and Methods Tears and peripheral blood samples were collected from patients with seasonal allergic conjunctivitis (SAC, n=6), vernal keratoconjunctivitis (VKC, n=12), and normal subjects (CT, n=12). From an additional six nonactive allergic patients, tears were collected before and after specific conjunctival allergen challenge (CAC). Upper tarsal conjunctival biopsies were obtained from five CT and five VKC patients. TNF-α in tears was measured by enzyme-linked immunoassay and identified in tissues by immunohistochemistry. Results Tear TNF-α levels in VKC patients were significantly increased compared to CT (p=0.03), and were significantly correlated with the severity of the disease. No differences were found between SAC and CT tear samples. TNF-α serum levels were higher in VKC than CT; however, this difference was not statistically significant. After CAC, tear TNF-a levels were found increased in only one of six patients. In VKC tissues, TNF-α positive cells were significantly increased compared to CT (p=0.03). Conclusions TNF-α may have a significant role in severe forms of allergic conjunctivitis.

Histamine-induced cytokine production and ICAM-1 expression in human conjunctival fibroblasts.

Purpose. Conjunctival fibroblasts stimulated with histamine (H) may be directly involved in the inflammatory and remodeling processes of chronic allergic conjunctival diseases. Methods. Proinflammatory cytokine and growth factor production, and the expression of intercellular adhesion molecule-1 (ICAM-1) were studied in conjunctival fibroblast cultures challenged with different concentrations of H (from 10 -9 M to 10 -4 M). Interleukin (IL)-1, IL-4, IL-6, IL-8, tumor necrosis factor-alfa (TNF-a), fibroblast growth factor (FGF), epidermal growth factor (EGF) and transforming growth factor-beta (TGFß-1) were measured in supernatants. ICAM-1 expression was evaluated by a fluorescence activated cell sorter (FACS). Inhibitory effects of the H-1 antagonists (antiH): emedastine, levocabastine, and azelastine, and of the antiH-2, cimetidine, on H-stimulated fibroblasts were evaluated by measuring both cytokines in supernatants and the cellular expression of ICAM-1. Results. Histamine increased the production of IL-1, IL-6 and IL-8, and ICAM-1 expression. TNF-a, IL-4 and growth factor production were not modified by histamine. The antiH-1, emedastine, significantly reduced H-induced production of IL-1, IL-6 and IL-8, while azelastine reduced only IL-1. Levocabastine and cimetidine were less effective. The histamine-induced increase in ICAM-1 expression was inhibited by emedastine but not by azelastine and levocabastine. Conclusions. Histamine has pro-inflammatory effects on conjunctival fibroblasts, inducing the production of cytokines and the expression of ICAM-1. Emedastine significantly reduced cytokine and ICAM-1 expression from H-stimulated fibroblasts. Conjunctival fibroblasts may contribute to the maintenance of inflammation in chronic allergic diseases.

Efficacy and safety of desonide phosphate for the treatment of allergic conjunctivitis.

Purpose. To study the safety and efficacy of the topical corticosteroid, desonide 0.25% ophthalmic solution, for inhibition of the clinical allergic reaction induced by conjunctival provocation (CPT) and for the treatment of seasonal allergic conjunctivitis (SAC). Methods. For the CPT study, 12 allergic but inactive patients were exposed in both eyes to increasing doses of a specific allergen until a positive bilateral, symmetrical early- and late-phase reaction was obtained. After 2 weeks the last positive dose was readministrated and their positive response confirmed. After an additional 2 weeks, CPT was performed 30 minutes after topical administration of desonide in one eye and placebo in the contralateral eye (Group A) or after topical desonide or placebo four times a day for 2 days (Group B). Clinical signs and symptoms were recorded after 15, 30, and 60 minutes, and after 6 hours. Regarding the seasonal study, 96 patients with active SAC were treated bilaterally with either desonide or fluorometholone for 3 weeks, and allergic signs and symptoms evaluated at regular intervals. The safety of the drugs was assessed by identification of any side effects or adverse events of any kind. Results. For the CPT study: individual itching and redness, and the sum score for signs and symptoms were all statistically (p < 0.05) and clinically (greater than 1 change between treated eyes) significantly lower in desonide versus placebo eyes. Both early- and late-phase reactions were reduced by desonide pretreatment. Seasonal study: desonide and fluorometholone were both highly effective in reducing itching, tearing, and conjunctival hyperemia over time (p < 0.0001). Both drugs appeared safe, with no statistically significant changes in IOP observed with either treatment. Conclusions. Desonide has a significant therapeutic effect on both the induced conjunctival early- and late-allergic reaction and in active SAC. It was also safe, with no side effects such as increases in intraocular pressure observed by physician or patient.

Effect of ofloxacin and netilmicin on human corneal and conjunctival cells in vitro.

The purpose of this study was to compare the cytotoxic effects of the fluoroquinolone ofloxacin with that of the aminoglycoside netilmicin. Human corneal epithelial cells (HCE-T) and human conjunctival epithelial cells (Wong-Kilbourne derivative of Chang conjunctiva) were exposed to antibiotics (0.08-5.0 mg/mL) for 4 or 24 hours. Cell proliferation and viability were assessed with the MTT assay, neutral red uptake, and bromo deoxy uridine incorporation. In both cell lines, ofloxacin inhibited cell proliferation and viability. These effects were time and dose dependent. Concentrations of ofloxacin ranging from 0.4 to 2.4 mg/mL (0.04% to 0.24%) produced a 50% inhibition of proliferation and viability. In contrast, netilmicin induced no toxic effect. The differences between ofloxacin and netilmicin were highly statistically significant (p < 0.001). This finding is particularly relevant in deciding the optimal antibiotic to be applied in clinical situations in which the epithelium is compromised.

Histology of ocular late-phase reaction in guinea pigs passively sensitized with IgG1 antibodies

The ocular late-phase reaction (LPR) is a mast cell-dependent, delayed inflammatory reaction developing 4-12 h after the early-phase reaction (EPR). We developed a passive IgG1 antibody-dependent guinea pig model that clinically reproduced the biphasic reaction of ocular EPR and LPR. An EPR was observed in all animals; a biphasic, multiphasic or prolonged inflammation was observed in the animals maintained for 9 and 24 h. The substantia propria of eyes undergoing EPR (0.5 h) showed intense edema, mast cell degranulation (88%), and 4-fold increase in eosinophils. At 9 h, the neutrophils and eosinophils had increased 11- and 25-fold, respectively. The number of basophils and lymphocytes was significantly increased compared to the controls (p less than 0.05). Of the mast cells, 45% were degranulated compared with 19% for controls. Cellular reactions had subsided by 24 h. Conjunctival epithelium also accumulated inflammatory cells as did the stroma of the lid skin. These histologic changes in ocular tissues undergoing anaphylaxis demonstrated that mast cell degranulation was most severe in the early phase, whereas neutrophil, eosinophil, and basophil accumulation was most marked in the late phase.

In vitro effects of fluoroquinolone and aminoglycoside antibiotics on human keratocytes.

Purpose: The purpose of this study was to assess the cytotoxic effects of the fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human keratocytes in vitro. Methods: Cultured human keratocytes were exposed to various concentrations of ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay) and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12, and 24 hours of incubation. Measurement of annexin V binding performed in association with the dye exclusion test using propidium iodide (PI) was also performed by FACS analysis after 4 hours of exposure. Results: Both antimicrobials induced dose- and time-dependent morphologic changes in keratocytes, yet the effects of netilmicin were minimal. After 24 hours of exposure, both drugs induced a dose-dependent inhibition of cell proliferation; however, ofloxacin demonstrated significantly more toxic effects than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus netilmicin, ANOVA, P < 0.05). Conclusions: Relative effects of aminoglycosides and fluoroquinolones on stromal keratocytes appear to be different: netilmicin was shown to be significantly less toxic than ofloxacin. This finding is particularly relevant in deciding the optimal antibiotic to be applied in clinical situations in which the epithelium is absent or compromised, as after photorefractive keratectomy, alkali burns, or ulcerative keratitis.

Clinical patterns of ocular anaphylaxis in guinea pigs passively sensitized with IgG1 antibody

A model of topically induced ocular anaphylaxis in guinea pigs was developed. A guinea pig anti-dinitrophenyl (DNP) bovine gamma-globulin antiserum rich in IgG1 antibodies was injected subconjunctivally; various serum dilutions and latent periods were tested. The anaphylactic response was elicited with di-DNP-lysine applied topically. The response was characterized by rating 11 ocular signs. Although the 4-hour latent period is considered optimal for passive cutaneous anaphylaxis, a 14-hour latent period yielded the strongest ocular reaction and appeared to the most appropriate time for clinical assessment. Both an early-phase reaction (peaking in the 21st hour) and a late-phase reaction (peaking at 6-10 h) were observed. The two signs of the early-phase reaction that recurred in the late-phase reaction were periorbital swelling and lid redness. The anaphylactic reaction displayed different patterns in different animals: protracted, biphasic and multiphasic.