A. Leonardi | Università degli Studi di Padova (original) (raw)

Papers by A. Leonardi

Journal of Autoimmunity, 1992

Cyclosporine-A should be active in different infla~atory conditions of the external eye, where T-... more Cyclosporine-A should be active in different infla~atory conditions of the external eye, where T-cell mediated immunopathologies play a significant role. In these conditions also a topical preparation of the drug should be effective, because of its penetrability through the conjunctiva and the external layers of the cornea. Our report deals with the excellent results we have obtained by treating with topical Cyclosporine-A (2% in castor oil) a group of patients affected by vernal keratoconjunctivitis.

Allergy, 2011

While it is known that histamine is the primary mediator of ocular allergy, the presence and dist... more While it is known that histamine is the primary mediator of ocular allergy, the presence and distribution of histamine receptors are not well documented in the human eye. Our aim was to evaluate histamine receptor expression in normal and vernal keratoconjunctivitis conjunctiva. Mucosal biopsies were obtained from conjunctiva of healthy donors and from tarsal conjunctiva of vernal patients. Immunostaining and semi-quantitative reverse-transcriptase polymerase chain reaction for H(1), H(2), H(3), and H(4) receptors were performed. Histamine receptor expression was also evaluated in conjunctival cell cultures exposed to histamine, interleukin-4, interleukin-5, interferon-γ and tumor necrosis factor-α. Immunostaining for H(1) and H(2) receptors was slightly positive in normal and over-expressed in vernal tissues. H(3) receptors were rarely present in normal and inflamed conjunctiva. In striking contrast to control tissues, H(4) receptors were highly expressed in all inflamed tissues, particularly by stromal inflammatory cells. Semi-quantitative reverse-transcriptase polymerase chain reaction demonstrated an over-expression of H(1), H(2), and H(4) receptors in vernal vs control tissues. Notably, H(4) receptors were five times more expressed in vernal vs control tissues. In cell cultures, H(2) receptor expression was stimulated eight times the normal levels by interleukin-4 and three times by histamine, but the H(4) receptor was only slightly affected by stimulation with these mediators. Increased expression of H1, and particularly of H(2) and H(4) receptors in vernal keratoconjunctival tissues indicate their important role in the pathogenesis of this disease. H(4) receptors may be a target in the treatment of allergic inflammation.

Experimental Eye Research, 1998

To quantify the presence of inflammatory\fibrogenic cytokines and procollagens type I (PICP) and ... more To quantify the presence of inflammatory\fibrogenic cytokines and procollagens type I (PICP) and III (PIIIP) in active and non-active tarsal and limbal forms of vernal keratoconjunctivitis (VKC), tear and blood samples were collected from 27 VKC patients (20 active and 7 non-active) and 15 normal subjects. Upper tarsal conjunctival biopses were obtained from 8 controls and 8 tarsal VKC patients. From biopses of 4 tarsal VKC, fibroblasts were cultured in F12 medium with 10 % FCS. TGF-β1, IL-1α, IL-1β, IL-6 and TNF-α, PICP and PIIIP were measured in : (1) tears, (2) homogenized conjunctival tissues, (3) serum, (4) supernatants of tissue cultures at 24 hr, and fibroblast primary passage cultures. Results showed : (1) in tears, TGF-β1 and TNF were identified in several active VKC patients without significant differences between the tarsal and the limbal forms. IL-1β (27p51 pg ml −" , P l 0n03) and IL-6 (28p43 pg ml −" , P l 0n006) were significantly increased in tarsal VKC compared to controls. Both control and non-active VKC tear samples had undetectable levels of all of the above cytokines. PICP and PIIIP were significantly increased in tarsal VKC compared to both limbal VKC and controls. Non-active VKC levels were similar to controls. (2) In homogenized VKC tissues, TGF-β1 and IL-6 were both significantly increased compared to controls (P 0n01) while no increases were observed in IL-1 and TNF-α. (3) In serum, IL-1α, IL-1β and TNF-α were higher in VKC patients compared to controls. (4) In vitro fibroblasts from VKC patients showed an increased production of TGF-β1, IL-1α, IL-1β, IL-6, TNF-α, PICP, and PIIIP over time. Increased levels of TGF-β1, IL-1 and IL-6 in VKC tissues and tears indicate a local production of these cytokines in active VKC. Collagen hyperproduction occurs only in active tarsal VKC and may be related to high levels of TGF-β1, IL-1 and IL-6. Increased serum levels of IL-1 and TNF-α suggests that systemic immunological changes occur in VKC. Cell culture can be used as a model to further study the pathogenesis of VKC and its characteristic local fibroblast activation.

Current Eye Research, 2002

Conjunctival fibroblasts stimulated with histamine (H) may be directly involved in the inflammato... more Conjunctival fibroblasts stimulated with histamine (H) may be directly involved in the inflammatory and remodeling processes of chronic allergic conjunctival diseases. 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-alpha), fibroblast growth factor (FGF), epidermal growth factor (EGF) and transforming growth factor-beta (TGFbeta-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. Histamine increased the production of IL-1, IL-6 and IL-8, and ICAM-1 expression. TNF-alpha, 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. 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.

British Journal of Ophthalmology, 2008

To determine the prevalence of vernal keratoconjuntivitis (VKC) in Europe. A cross-sectional surv... more To determine the prevalence of vernal keratoconjuntivitis (VKC) in Europe. A cross-sectional survey was mailed to 3003 ophthalmologists from six countries (Finland, France, Italy, The Netherlands, Norway and Sweden) representing 151.9 million inhabitants. Results were analysed per country, and VKC prevalence for the 15 European member states in 2002 was extrapolated. Six hypotheses were used: disease duration (4 or 8 years) combined with three prevalence hypotheses for non-responding ophthalmologists. The response rate to the survey was 29.5%. The estimates of VKC prevalence in Western Europe (per 10,000 inhabitants) ranged from 1.16 to 10.55. The prevalence of VKC with corneal complications ranged from 0.30 to 2.26. The VKC prevalences per country were in the following ranges: Italy 2.4-27.8, Finland, 0.7-8.4, Sweden 1.2-8.7, The Netherlands 0.6-4.6, France 0.7-3.3 and Norway 0.3-1.9. VKC with corneal complications were: Italy 0.4-4.8, Sweden 0.3-2.4, Finland 0.2-2.8, The Netherlands 0.2-1.6, France 0.3-1.4 and Norway 0.1-1.0. Based on the most likely hypotheses concerning disease duration and non-responding ophthalmologists&amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; VKC case rate, the best estimate of VKC prevalence in Western Europe is 3.2/10,000 inhabitants. The prevalence of VKC with corneal complications is 0.8/10,000 inhabitants.

Investigative ophthalmology & visual science, 2000

To study the extracellular composition of giant papillae in vernal keratoconjunctivitis (VKC) and... more To study the extracellular composition of giant papillae in vernal keratoconjunctivitis (VKC) and the expression of growth factors that may stimulate fibrosis. Upper conjunctival specimens were obtained by biopsy in 9 patients affected by active tarsal VKC (14 eyes) and 10 normal control subjects. Immunohistochemistry was performed on tissue sections using monoclonal antibodies (mAbs) for collagens I, III, and VII; tumor necrosis factor (TNF)-alpha; transforming growth factor (TGF)-ss1; basic fibroblast growth factor (bFGF); and platelet-derived growth factor (PDGF). The mAbs anti-tryptase, anti-CD4, anti-CD68, and anti-EG2 were used as markers for mast cells, T-helper lymphocytes, macrophages, and eosinophils, respectively. Immunofluorescent double-staining for growth factors and cell markers was performed in VKC tissues. Immunostaining was highly positive for collagens I, III, and VII in the subepithelium of VKC conjunctiva. Image analysis showed a significant increase of staining...

Investigative Opthalmology & Visual Science, 2003

PURPOSE. To determine whether cytokines involved in chronic allergic conjunctival disorders may a... more PURPOSE. To determine whether cytokines involved in chronic allergic conjunctival disorders may affect formation of giant papillae and tissue remodeling. METHODS. Conjunctival fibroblast cultures were challenged with different concentrations of human recombinant interleukin (IL)-4, IL-13, interferon (IFN)-␥ and tumor necrosis factor (TNF)-␣. Procollagens I (PIP) and III (PIIIP), matrix metalloproteinase (MMP)-1 and-9, and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants, and their respective mRNAs were evaluated by RT-PCR. RESULTS. IL-4 and-13 (10 ng/mL) significantly increased production and expression of PIP compared with nonstimulated cells, whereas IFN-␥ elicited the opposite effect, at both the protein and mRNA levels. Both IL-4 and-13 significantly decreased production of MMP-1 and increased that of TIMP-1, whereas TNF-␣ increased production of MMP-1 and-9. Expression of MMP-1 was reduced by IL-4 and increased by the other tested cytokines, whereas expression of TIMP-1 was increased by all tested cytokines. CONCLUSIONS. IL-4 and-13 increased production of collagen and modified the equilibrium between MMP-1 and its inhibitor, TIMP-1. These effects were partially opposed by IFN-␥ and TNF-␣.

Investigative Opthalmology & Visual Science, 2005

PURPOSE. Plasminogen activators play a role, not only in fibrinolysis but also in events such as ... more PURPOSE. Plasminogen activators play a role, not only in fibrinolysis but also in events such as chemotaxis, collagen degradation, and cell spreading. The serine protease urokinase (uPA) is a potent chemoattractant for leukocytes that may be involved in the pathogenesis of severe forms of allergic conjunctivitis such as vernal keratoconjunctivitis (VKC). METHODS. Tear and peripheral blood samples were obtained from 20 patients with active VKC and from 19 normal subjects who formed the control group. Levels of plasminogen activity, uPA, tissue plasminogen activator (tPA), and their inhibitor, plasminogen activator inhibitor type-1 (PAI-1) were measured in tears and plasma of patients with VKC. The presence of tPA, uPA, and urokinase receptor (uPAR) in conjunctival tissues were evaluated by immunohistochemistry. uPA, uPAR, and PAI-1 expression and production were measured in conjunctival epithelial cell and fibroblast cultures treated with cytokines. RESULTS. Tear levels of uPA and tPA and tear plasminogen activity levels were significantly greater in patients with VKC than in control subjects. Increased staining for uPA and uPAR was found in VKC tissues compared with normal conjunctiva. Both conjunctival epithelial cells and fibroblasts demonstrated an increased expression of uPAR after exposure to IL-4 or-13, whereas uPA was highly expressed by epithelial cells exposed to IL-4. PAI-1 levels in culture medium were increased in IL-4-exposed epithelial cells compared to nonstimulated cells and were decreased in fibroblast culture. CONCLUSIONS. Increased expression of fibrinolytic system components and imbalance between plasminogen activators and PAI may be involved in the pathogenesis of severe allergic conjunctivitis, thus contributing to inflammatory cell migration and tissue remodeling.

Investigative ophthalmology & visual science, 1999

Th2 lymphocytes may play a key role in the development of allergic diseases such as vernal kerato... more Th2 lymphocytes may play a key role in the development of allergic diseases such as vernal keratoconjunctivitis (VKC). Cytokine flow cytometry of tear samples was used to identify the phenotypical and functional properties of lymphocytes at the actual site of the allergic reaction. Tear and blood samples were obtained from patients affected by active VKC (n = 12) and from normal control subjects (n = 10). Tears were obtained after gentle scraping of the tarsal and bulbar conjunctiva. Tear and blood samples were placed in a solution of brefeldin-A, phorbol myristate acetate (PMA), ionomycin, and RPMI for 4 hours and then processed for flow cytometry. Lymphocytes were marked with the monoclonal antibodies, anti-IFN-gamma and anti-interleukin (IL)-4. Levels of IL-4, IL-2, IFN-gamma, IL-2R, total IgE, eosinophil cationic protein (ECP), eosinophil protein X/neurotoxin (EPX), and myeloperoxidase (MPO) were also evaluated in serum. Expression of IL-4 was observed in 9.2%+/-9.5% of lymphocy...

Investigative ophthalmology & visual science, Jan 22, 2015

Purpose: Wilson disease (WD) is a disorder of hepatic copper metabolism leading to copper accumul... more Purpose: Wilson disease (WD) is a disorder of hepatic copper metabolism leading to copper accumulation in hepatocytes and in extrahepatic organs, as the brain and the cornea. The aim of this study was to investigate central corneal changes and in particular to assess the parameters of corneal sub-basal nerve plexus (SBNP) in patients affected by WD, using corneal confocal microscopy (CCM). Twenty-four patients affected by WD and 24 healthy control subjects were incuded in this cross-sectional comparative study. One eye of each subject was examined to quantify different corneal parameters. Mean cell diameter and mean cell density of the epithelium; number of fibers (NF), nerve fiber length density (NFLD), number of branchings (NBr), number of beadings (NBe) and fiber tortuosity (FT) of the SBNP; mean cell density of keratocytes of the anterior, medium and posterior stroma and mean cell density, polimegatism and pleomorphism of the endothelium and central corneal sensitivity were anal...

Experimental Eye Research, 1992

Clinical <html_ent glyph="@amp;" ascii="&"/> Experimental Allergy, 2006

Background Several cytokines are involved in the recruitment and activation of inflammatory cells... more Background Several cytokines are involved in the recruitment and activation of inflammatory cells in ocular allergic diseases. The purpose of the study was to assay multiple cytokines and chemokines in tears, to compare subgroups of allergic conjunctivitis (AC) with controls, and in culture supernatants to determine whether conjunctival fibroblasts produce some of these cytokines. Methods Fifty to one hundred microlitre tears were obtained from patients with active seasonal allergic conjunctivitis (SAC; n = 12), vernal keratoconjunctivitis (VKC; n = 18), atopic keratoconjunctivitis (AKC; n = 6) and non-atopic controls (n = 14). Primary conjunctival fibroblasts grown in vitro were stimulated with IL-4, IL-13 or TNF-a for 24 h. Cell-free tear and culture supernatants were assayed for IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IFN-g, TNF-a, eotaxin, MCP-1 and RANTES using multiplex bead analysis. Induction of chemokine gene expression was determined by PCR. Results IL-1b, IL-2, IL-5, IL-6, IL-12, IL-13, MCP-1 were increased in all tears groups compared with controls, with highly significant correlations between many of these molecules. In addition IL-4, IFN-g, and IL-10 were elevated in SAC and VKC, while eotaxin and TNF-a were only increased in VKC. IL-6, IL-8, MCP-1, RANTES and eotaxin were detected from fibroblasts cultures, and were all up-regulated by TNF-a. By PCR, fibroblasts expressed MCP-1 transcripts constitutively, whereas IP-10 and Mig were up-regulated by TNF-a. Conclusions Differential cytokine levels support tears as a useful indicator of immune mechanisms occurring during AC. The striking similarities in chemokine profiles between tears and fibroblasts suggest these cells as likely sources of chemokines in tears.

Clinical & Experimental Allergy, 2010

Background Vernal keratoconjunctivitis (VKC) is a chronic ocular allergic inflammation characteri... more Background Vernal keratoconjunctivitis (VKC) is a chronic ocular allergic inflammation characterized by corneal complications and the formation of giant papillae. Sma-and Madrelated proteins (Smad) modulate extracellular matrix gene expression during wound healing, inflammation and tissue remodelling. Objective To investigate the relationship between allergic inflammation and TGF-b/Smad signalling pathway, expression in VKC patients and in primary cultured conjunctival fibroblasts exposed to mediators found previously over-expressed in VKC. Methods Smad-2,-3,-7, phospho-(p)Smads, TGF-b1 and-b2 were evaluated in the conjunctiva of normal subjects (CT) and VKC patients by immunohistochemistry. The expression of Smads, pro-collagen I (PIP), TGF-b1,-b2, mitogen-activated protein kinase (p38/MAPK), c-Jun Nterminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2) were also determined in conjunctival fibroblast cultures exposed to histamine, IL-4,-13, TGF-b1, IFN-g and TNF-a using immunostaining or RT-PCR. Results Immunostaining for Smad-2,-3, pSmad-2,-3, TGF-b1,-b2 and PIP was significantly increased in VKC stroma compared with CT. In conjunctival fibroblast cultures, Smad-3 and PIP were stimulated by histamine, IL-4,-13 and TGF-b1 exposure, while PIP was reduced by IFN-g, and TNF-a mRNA expression of Smad-3 was increased by histamine, while Smad-7 was reduced by IL-4. In addition, histamine, IL-4 and TNF-a increased JNK and ERK1/2 expression. Conclusion and Clinical Relevance The TGF-b/Smad signalling pathway is over-expressed in VKC tissues and modulated in conjunctival fibroblasts by histamine, IL-4, TGF-b1 and TNF-a. These mechanisms may be involved in fibrillar collagen production, giant papillae formation and tissue remodelling typical of VKC and might provide new therapeutic targets for its treatment.

British Journal of Ophthalmology, 2000

Aim-To study the eVect of the topical anti-inflammatory drug, ketorolac, on (1) the clinical alle... more Aim-To study the eVect 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 diVerence in the epithelial expression of ICAM-1 was observed between placebo and ketorolac treated eyes (p<0.05). Conclusion-Ketorolac proved to be eVective in reducing mast cell degranulation, as indicated by significantly decreased tryptase tear levels, as well as the clinical and cytological allergic reaction.

British Journal of Ophthalmology, 1995

Aims-The objective of this study was to investigate alterations in conjunctival collagen and prot... more Aims-The objective of this study was to investigate alterations in conjunctival collagen and proteoglycans in the conjunctival giant papillae of patients with vernal keratoconjunctivitis (VKC). Methods-Tissue samples from tarsal giant papillae of seven eyes from five patients with VKC, and five tarsal conjunctival samples from five normal patients were obtained. Tissues were processed and stained with haematoxylin and eosin, Van Gieson, trichromic Mallory, toluidine blue, Alcian blue, and alkaline Giemsa. Collagen extraction was performed in acetic acid and pepsin, total collagen was quantified using hydroxyproline levels, and collagen types I and III were analysed by gel electrophoresis (SDS-PAGE). Proteoglycans were quantified using uronic acid levels. Results-Histological evaluation showed a significant increase of mast cells in the epithelium (0/mm2 v 147/mm2, p<0.01) and in the stroma (5.1/mm2 v 80/mm2, p<0-Ol) of VKC patients. Collagen fibres were thicker and arranged irregularly, with the total amount significantly increased. Owing to an increased percentage of type III collagen, the ratio of collagen types I to III was decreased. Proteoglycans were also reduced in VKC samples. Conclusion-The well known morphological abnormalities observed in VKC correspond to alterations in the ratio between collagens and proteoglycans, and between different types of collagen. The greatly increased number of mast cells found in these tissues suggests an active role for these cells in the abnormal connective tissue metabolism observed in VKC.

European journal of ophthalmology

To quantify the presence of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) ... more To quantify the presence of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in allergic conjunctivitis. 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-alpha in tears was measured by enzyme-linked immunoassay and identified in tissues by immunohistochemistry. Tear TNF-alpha 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-alpha serum levels were higher in VKC than CT, however, this difference was not statistically significant. After CAC, tear TNF-...

American Journal of Ophthalmology, 1990

We treated 11 patients with vernal keratoconjunctivitis for four to nine months with topical cycl... more We treated 11 patients with vernal keratoconjunctivitis for four to nine months with topical cyclosporine as a 2% dilution in castor oil. No significant side effects occurred, except for mild and transient burning upon administration. Within the first 15 days, both symptoms and signs of the condition improved significantly, and these results were maintained throughout the entire treatment. Relapses of the disease occurred two to four months after the end of the therapy. A double-masked clinical trial of nine patients (2% cyclosporine in castor oil vs castor oil alone) confirmed the results. Treated eyes improved significantly for both signs and symptoms as compared to control eyes. Topical cyclosporine may, therefore, be considered an effective substitute for corticosteroids, with an excellent anti-inflammatory activity in patients with both corticosteroid-dependent and corticosteroid-resistant vernal keratoconjunctivitis.

Acta Ophthalmologica Scandinavica, 1999

ABSTRACT

Acta Ophthalmologica Scandinavica, 2000

To compare emedastine ophthalmic solution 0.05% BID to levocabastine ophthalmic suspension 0.05% ... more To compare emedastine ophthalmic solution 0.05% BID to levocabastine ophthalmic suspension 0.05% BID in reducing chemosis, eyelid swelling and other signs and symptoms in subjects with seasonal allergic conjunctivitis. In a randomized, double-masked, parallel controlled study, emedastine ophthalmic solution 0.05% BID was compared to levocabastine ophthalmic suspension 0.05% BID for control of chemosis, eyelid swelling and other parameters in the environmental allergy study model. At Days 7, 14, 30 and 42, emedastine was significantly better than levocabastine at controlling chemosis and eyelid swelling (p < 0.05). A statistical trend was seen at Day 3 (0.05 < p < 0.10). Results were clinically relevant at Days 30 and 42. Emedastine was also significantly better at reducing redness and itching at Days 7, 14, 30 and 42 (p < 0.05). Emedastine is more efficacious than levocabastine in reducing chemosis, eyelid swelling and other efficacy variables associated with seasonal allergic conjunctivitis.