Antonio G. Secchi

Topical Use of Cyclosporine in the Treatment of Vernal Keratoconjunctivitis

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.

Tear and serum soluble leukocyte activation markers in conjunctival allergic diseases.

PURPOSE To measure markers of leukocyte activation in patients with an exclusively ocular inflammatory or bacterial disease. METHODS Neutrophil myeloperoxidase, eosinophil cationic protein, eosinophil neurotoxin, and soluble interleukin-2 receptor were measured in serum and tears of 17 patients with allergic vernal keratoconjunctivitis, seven with atopic keratoconjunctivitis, 11 with seasonal allergic conjunctivitis, seven with giant papillary conjunctivitis, 13 with rosacea blepharokeratoconjunctivitis, seven with bacterial conjunctivitis, and 13 normal subjects as controls. RESULTS In serum of patients with vernal and atopic keratoconjunctivitis, levels of eosinophil cationic protein, eosinophil neurotoxin, and interleukin-2 receptor were significantly increased compared with control subjects but were not correlated with the severity of ocular symptoms. In tears of patients with vernal and atopic keratoconjunctivitis and seasonal allergic conjunctivitis, as well as in the nonallergic diseases, rosacea blepharokeratoconjunctivitis and bacterial conjunctivitis, levels of eosinophil cationic protein, neurotoxin, and interleukin-2 receptor were significantly increased compared with control subjects. The highest values of these markers were found in vernal keratoconjunctivitis samples. Neutrophil myeloperoxidase was significantly increased in vernal and atopic keratoconjunctivitis, rosacea blepharokeratoconjunctivitis, and bacterial conjunctivitis. In vernal keratoconjunctivitis, tear markers were correlated to the clinical score of the disease, but not with cytology. CONCLUSIONS Tear histamine was measured in 10 allergic patients after allergen challenge. Although none of the above markers can be considered specific to a single disease, their measurement may still be useful for the quantification of local cell activation in ocular inflammatory diseases.

Histaminase Activity in Patients with Vernal Keratoconjunctivitis

PURPOSE To investigate the activity of histamine-degradating enzymes in tears and plasma of patients with vernal keratoconjunctivitis (VKC). METHOD Tear and plasma samples were collected from patients with VKC and from age-matched control subjects. Histamine was measured by enzyme-linked immunosorbent assay in acid samples treated with perchloric to deactivate histaminase and in untreated samples. Tear cytology, skin test reactivity to histamine, and the sum clinical score of allergic signs and symptoms in patients with VKC also were evaluated. Nineteen patients with active VKC and six age-matched control subjects participated in this study. RESULTS In untreated samples, tear histamine (mean +/- standard error of the mean) was 11.15 +/- 2.16 ng/ml in patients with VKC and 0.855 +/- 0.225 ng/ml in control tears (P < 0.001). In treated samples, mean tear histamine was 22.25 +/- 4.17 ng/ml in patients with VKC versus 10.64 +/- 2.85 ng/ml in control subjects (not statistically different). The ratio of histamine in treated to untreated samples (indicating histaminase activity) was significantly lower in patients with VKC (2.30 +/- 0.263) than in control subjects (17.57 +/- 5.97; P = 0.0001). Plasma histamine levels in untreated and treated samples were significantly higher in patients with VKC (untreated, 2.23 +/- 0.334 ng/ml; treated, 4.37 +/- 0.357 ng/ml) than in control subjects (untreated, 0.254 +/- 0.068, P = 0.0002; treated, 2.96 +/- 0.171 ng/ml, P = 0.0082). The enzymatic breakdown of histamine (treated/ untreated) in plasma was significantly decreased in patients with VKC (2.54 +/- 0.447) compared with control subjects (14.78 +/- 4.86; P = 0.0012). Skin reactivity to histamine was not increased in VKC. Tear histamine levels were significantly correlated to tear lymphocyte content in the general population and to tear basophils in the patients with tarsal-vernal VKC only. An increased number of tear eosinophils were correlated with elevated enzyme activity only in patients with tarsal-vernal VKC and to the clinical score only in limbal-vernal patients. CONCLUSION The enzymatic degradation of histamine was significantly decreased in patients with VKC compared with control subjects in both tears and plasma, suggesting that this dysfunction may be a primary factor in the pathophysiology of VKC.

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.

Antigen sensitivity evaluated by tear-specific and serum-specific IgE, skin tests, and conjunctival and nasal provocation tests in patients with ocular allergic disease

The potential for ocular allergic patients to have a site-specific antigen sensitisation was investigated using various diagnostic tests of allergen sensitivity in subjects with allergic conjunctivitis (AC: n = 135), vernal keratoconjunctivitis (VK: n = 20), rhinoconjunctivitis (n = 20) or rhinitis (N = 10). In the AC and VK patients, skin tests and conjunctival provocation tests (CPT) were performed, and the levels of specific IgE in serum and in tears were identified. A subgroup of 36 patients was also challenged with a nasal-specific provocation test (NPT). Results showed a poor correlation between skin test results and tear-specific IgE, and also between serum-specific IgE and tear-specific IgE in both AC and VK patients (K<0.3). CPT and tear IgE were significantly correlated (K = 0.5) in the ocular allergic population. In patients with rhinoconjunctivitis or rhinitis, and in 10 normal subjects, results of CPT and NPT were in 100% agreement. Conversely, in patients with only conjunctivitis, little correlation was found between the results of CPT and NPT (K = 0.3). Tear-specific IgE was the only positive diagnostic sign of antigen sensitivity in 35% of VK patients and 30% of AC patients. These results suggest that the conjunctiva can be a uniquely sensitised target organ in allergic patients.

Collagen types I and III in giant papillae of vernal keratoconjunctivitis.

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 hydroxy-proline 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 < 0.01) 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.

Cyclosporine-A in the treatment of serpiginous choroiditis

SummarySeven patients affected by bilateral inflammatory serpiginous choroiditis have been treated with Cyclosporine-A for 6-21 months. Nine outof the fourteen eyes showed a significant improvement in their visual acuity; five eyes did not change.Cyclosporine-A may, therefore, be considered effective in the treatment of this disease.Its usefulness seems to be greater when the serpiginous choroiditis is in its ‘acute’ stage; ‘chronic’ stages, however, also seem to improve under treatment.Its main indication is, in our opinion, the involvement of the macular region of the ‘second’ eye, when the ‘first’ eye is already damaged. We consider Cyclosporine-A, in these situations, to be a first choice treatment.

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.

Correlation Between Conjunctival Provocation Test (CPT) and Systemic Allergometric Tests in Allergic Conjunctivitis

In order to assess the potential usefulness of CPT as a diagnostic tool for ocular allergy, the correlation between skin/RAST tests and CPT was determined in 144 patients affected by allergic ‘hay fever’ type conjunctivitis. The results showed that an agreement between skin/RAST tests and CPT occurred in 71% of the cases (130/ 183). Of the 29% uncorrelated cases, 23% (43/183) were positive for at least one specific antigen by skin/RAST tests but not by CPT, while 6% (10/183) were positive for at least one specific antigen by CPT, but not by skin/RAST tests. CPT dramatically increased the histamine levels in tears (p<0.001). These findings show that (1) systemic tests can be misleading in that they may suggest a specific sensitisation which, in fact, does not involve the conjunctiva (systemic test positive/CPT negative); (2) CPT can identify local conjunctival sensitisation in the absence of a systemic sensitisation (systemic test negative/CPT positive); (3) CPT can demonstrate that allergic ‘hay fever’ type conjunctivitis may be related to allergens different from those responsible for a systemic sensitisation.

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.