Cristiana Valente

Immune response in the conjunctival epithelium of patients with dry eye

The aim of our project was to test the hypothesis that patients with dry eye have a significant degree of inflammation and lymphocyte infiltration in conjunctival epithelium by using flow cytometry analysis of cells stored in cell culture medium. Impression cytology specimens were collected in 15 normal subjects and 15 dry eye patients. Samples collected from the right eye were placed in Phosphate Buffered Saline containing 0.05% paraformaldehyde (PFA), and samples from the left eye in cell culture medium containing 10% foetal calf serum (FCS). Phenotypic analysis was performed on cells derived from 21 dry eye patients and 16 healthy controls. The cells collected in FCS were stained for the expression of CK19, CD3, CD4, CD8, CD56, CD19, CD20, CD14 and HLA-DR, and analyzed by flow cytometry. FCS samples contained a statistically increased number of cells when compared to PFA samples. No statistically significant differences were present in the number of CD45+CK19- cells, CD3+ and CD4+T cells, B and NK cells in dry eye patients compared to healthy controls. In the dry eye group there was a significant difference in the CD4/CD8 ratio respect to what observed in normal subjects, and an increased number of CD14+ cells. HLA-DR expression was increased only in CK19+ conjunctival epithelial cells of dry eye patients. This study indicates that immune cells isolated from the superficial layer of the conjunctiva may play a pivotal role in the pathogenesis of dry eye, and that a new method of preservation of impression cytology samples can enhance flow cytometry analysis of epithelial and immune cells of the conjunctiva.

New test to quantify lipid layer behavior in healthy subjects and patients with keratoconjunctivitis sicca.

Purpose: Diagnostic procedures currently available to evaluate tear film lipid layer alterations are of limited application and do not provide quantitative results. The purpose of this prospective, case-control study was to develop a noninvasive test to measure tear lipid behavior in healthy subjects and patients with dry eye. Methods: The dynamic lipid layer interference patterns (DLIP) test was performed on 21 patients with dry eye and 21 age-matched controls. Subjects in the study and control groups were asked to perform 5 forced blinks and 10 consecutive nonforced blinks every 2 seconds to load lipids from the palpebral reservoir into the tear film. After recording the lipid layer interference patterns obtained with the Tearscope, a masked investigator counted the number of blinks to observe significant changes of shape, position, and number of waves of the interference patterns. Patients with dry eye were identified on the basis of the typical symptoms measured by a validated questionnaire (Ocular Surface Disease Index questionnaire score >10), Schirmer I test scores <10 mm/5 min, tear breakup time (TBUT) <7 seconds, and lissamine green conjunctival staining >4. Results: Significant differences in Schirmer test, TBUT, and lissamine green were recorded between groups. The DLIP test in the dry eye group (2.4 ± 3.1 blinks) was statistically decreased compared with the control group (18.1 ± 5.9 blinks; P < 0.0001, t test). A significant Pearson correlation (r = 0.788) was found between the DLIP test and TBUT. The receiver operating characteristic curve defined a cutoff value of 6.5 blinks to separate healthy from dry eyes (sensitivity of 100%, specificity of 95%). Conclusions: The DLIP is a new test that can be used in clinical practice to quantify tear film lipid layer behavior and to diagnose dry eye.

Symptoms and Signs of Tear Film Dysfunction in Glaucomatous Patients

PURPOSE The purposes of this study were to evaluate the presence of symptoms of tear film dysfunction by using the Ocular Surface Disease Index (OSDI) questionnaire in glaucomatous patients and to examine whether they have ocular surface signs. METHODS Fifty patients with ocular hypertension or open-angle glaucoma were sequentially examined. All patients used preserved antiglaucomatous drops once, twice, 3 times, or 4 times a day. Each patient filled out an OSDI questionnaire. Fluorescein corneal staining, lissamine green conjunctival staining, break-up time, and Schirmer I test were performed in patients with positive OSDI. RESULTS Using the OSDI, 26 of 50 patients (52%) showed at least mild symptoms of tear film dysfunction (score >12); of them, 17 (34%) had severe OSDI (Score >32). Decrease in tear production was seen in only 8 glaucomatous patients. The break-up time was <7 s in 18 patients. Lissamine green conjunctival staining was positive in at least one eye of all the 26 patients, whereas fluorescein corneal staining was positive in at least one eye of 22 patients. A statistically significant (P<0.05) difference was found for lissamine green conjunctival staining between 16 patients using ipotensive drops once or twice a day and 10 patients using drops 3 or 4 times a day. In the group of patients using only β-blocker agents, we found a positive correlation between symptoms and vital staining of the ocular surface. CONCLUSION Fifty-two percent of patients in therapy with preserved antiglaucomatous drops showed symptoms of tear film dysfunction. Signs of ocular surface diseases seemed to be greater in patients under >2 medications. Symptoms correlated to signs only in patients in monotherapy with β-blockers drops.

Anatomical and immunological changes of the cornea in patients with pterygium.

Purpose: To test the hypothesis that anatomical and immunological changes are present in the cornea of patients affected by pterygium. Materials and Methods: The central cornea of 20 eyes of 18 patients with primary pterygium was examined by in vivo confocal microscopy using a 40× lens and an axial resolution of 5 μ m. The size of pterygia was measured by analyzing photographic images. Data from 20 age-matched normal subjects were used as control for analysis. Results: The images obtained showed a significant lower number of epithelial cells in patients with pterygium compared to controls, with superficial epithelial cell area considerably higher than normal, reduced nucleus/cytoplasm ratio, halos around the nuclei, and sharp borders. Numerous highly reflective dendritic-like cells were present in the epithelial cell basal layer. The stroma showed a loss of keratocytes and the presence of lacunae. Tortuosity and number of vesicles of sub-basal nerves were significantly higher than controls. The endothelial cell count showed a normal density of cells of this layer. Conclusions: In vivo confocal microscopy may be helpful in evaluating the immunological and structural changes of the cornea in patients with pterygium and understanding its pathophysiology and the possible role of anti-inflammatory therapy.

Impact of glaucoma medication on ocular tissue

Open-angle glaucoma and ocular hypertension require chronic topical treatment whose active ingredients can have some local side effects. Furthermore, different preservatives are present in the bottles to avoid contamination by common pathogens. Among all the preservatives, benzalkonium chloride is the most common and its toxic effects on the ocular tissue are well known. The purpose of this article is to investigate the ocular effects of glaucoma medication on ocular tissue, and in particular the ocular changes caused by preserved antiglaucoma drugs. Moreover, the shift from preservative-containing to preservative-free drugs, or the use of alternative preservatives with a lighter toxicity could be useful to decrease the negative impact of glaucoma medications on ocular tissues and to improve the patient’s compliance. The outcome of filtering surgery could change on the basis of a previous topical therapy.