Tsutomu Fujihara

Conjunctival Epithelium Expression of HLA-DR in Dry Eye Patients

Purpose: To compare conjunctival epithelium expression of HLA-DR and ICAM-1 with tear dynamics and ocular surface parameters. Methods: Brush cytology and flow cytometry were used to quantitatively analyze HLA-DR and ICAM-1 expression in 28 dry eye patients. Results: HLA-DR was expressed in 66% of the conjunctival cells of dry eye patients. This expression correlates with that of ICAM-1, as well as with the Schirmer test results (p < 0.05). Conclusion: This study confirmed that dry eye consists not only of ocular surface desiccation, but also of upregulation of the HLA-DR and ICAM-1 molecule in the conjunctival epithelium, possibly resulting in increased inflammation.

INS365 Suppresses Loss of Corneal Epithelial Integrity by Secretion of Mucin-like Glycoprotein in a Rabbit Short-term Dry Eye Model

P2Y2 receptor agonists, like UTP and ATP, stimulate mucin secretion from goblet cells in vitro. Therefore, mucin stimulants could be good candidates for the treatment of dry eye syndrome because mucin increases the tear film stability and protects against desiccation of ocular surface. INS365 is a more stable P2Y2 receptor agonist than UTP. In the present study, we evaluated, in normal rabbit eyes, its effectiveness to release mucin from goblet cells and to protect the corneal damage induced by desiccation. For mucin secretion, impression cytology was performed following the instillation of INS365 solution or saline into the conjunctival sac. The specimens were stained with periodic acid and Schiff (PAS) reagent, and then the staining area was calculated using computer software. INS365 dose-dependently decreased the PAS staining area of conjunctival goblet cells from 2 to 15 min post-application. Furthermore, we utilized the rabbit short-term dry eye model to evaluate if INS365 eyedrops could protect against any of the damage produced by blockage of blinking with ocular speculum. INS365 significantly suppressed corneal damage at concentrations of more than 0.1% w/v. These results suggest that this P2Y2 agonist is a good candidate for the treatment of dry eye disease.

Diquafosol Elicits Increases in Net Cl– Transport through P2Y2 Receptor Stimulation in Rabbit Conjunctiva

The purpose of the present study was to understand the mechanisms of action of diquafosol, a stable derivative of uridine 5′-triphosphate, on Cl– transport across the isolated rabbit conjunctiva. Rabbit conjunctivas were isolated and mounted in a modified Ussing chamber. Under short-circuit conditions, the effects were determined of mucosal (tear) side diquafosol application on the short-circuit current (Isc). Diquafosol rapidly and dose-dependently increased the Isc at concentrations ranging from 0.1 to 968 µM when added to the mucosal side of the conjunctiva. In the absence of the serosal Cl–, the Isc induced by 10 µM diquafosol was substantially reduced. On the contrary, in the absence of mucosal side Na+, the diquafosol-induced increases in Isc were unchanged. Following 45-min preincubation, the P2Y2 antagonist suramin inhibited the diquafosol-induced increases in the Isc whereas the P2Y1 antagonist pyridoxal-phosphate-6-azophenyl-2′4′-disulfonic acid had no effect. These studies suggest that diquafosol stimulates net Cl– secretion from the serosal to the mucosal side via stimulation of P2Y2 receptors in the rabbit conjunctiva.

P2Y2 receptor stimulation increases tear fluid secretion in rabbits

Purpose. The purinergic P2Y 2 receptor agonists stimulate active Cl - transport across the excised rabbit conjunctival tissue in vitro. We determined whether UTP or ATP could increase the tear volume and change tear fluid composition in normal rabbits in vivo. Methods. Fifty µL was applied to rabbit eyes of UTP, ATP at concentrations of 0.1, 0.3, 1, 3, 8.5% (1.8–154 mM) or saline. A modified Schirmer test with topical anesthesia was performed 5, 15, 30 and 60 min after the instillation. In studies lasting 30 days, 50 µL of 0.5% UTP was applied 6 times a day for 4 weeks. Tear samples were collected from the conjunctival sac with a glass microcapillary. The protein profile of the tear fluid was analyzed by SDS-polyacrylamide gel electrophoresis and total protein was measured with the Bradford assay. The Easy-Titer rabbit IgG assay kit was used for the determination of immunoglobulin G (IgG). Results. UTP had dose-dependent stimulatory effects on tear secretion. It maximally increased tear secretion about 4-fold 15 min after its application. Similar effects were obtained with ATP. Repeated treatment with UTP reproducibly increased tear volume. Furthermore, UTP did not decrease total protein and IgG concentration in tear fluid and it had no effect on the protein profile. Conclusion. These data indicate that activation of P2Y 2 receptor increases tear fluid secretion accompanied with some proteins in normal rabbits. The purinergic agonists, UTP and ATP, have potential therapeutic value in the treatment of dry eye.

Diseases associated with ocular surface abnormalities: the importance of reflex tearing.

AIM To investigate the correlation between tear function tests and ocular surface integrity in patients with dry eye. METHODS 297 dry eye patients (55 Sjögren’s syndrome, two male and 53 female, average age 52.4 (SD 15.0) years, and 242 non-Sjögren’s syndrome, 41 male and 201 female, average age 53.5 (14.1) years) were examined. The following tear function tests were performed: (1) cotton thread test, (2) Schirmer test with topical anaesthesia, (3) Schirmer test without anaesthesia, (4) Schirmer test with nasal stimulation, (5) tear clearance test, and (6) tear break up time (BUT). The ocular surface was evaluated by rose bengal and fluorescein staining. Correlation analysis was performed between each tear function index and vital staining scores. RESULTS Among the six tear function tests, the Schirmer test with nasal stimulation correlated most with both of the vital stains (ρ=0.530 for rose bengal and 0.393 for fluorescein). The Schirmer test with or without anaesthesia correlated slightly with rose bengal staining, whereas tear clearance test and tear break up time slightly correlated with fluorescein staining. CONCLUSION Vital staining of the ocular surface correlates most with reflex tearing measured by the Schirmer test with nasal stimulation.

Flow Cytometric Analysis of Surface Antigens on Human Conjunctival Epithelial Cells

We analyzed the levels of expression of different surface adhesion molecules on normal human conjunctival epithelial cells with cytology and flow cytometry. The levels of beta 1 (CD29), VLA-1 (CD49a), VLA-2 (CD49b), and VLA-3 (CD49c) integrins were high, whereas those of VLA-5 (CD49e) and VLA-6 (CD49f) were faint, and that of VLA-4(CD49d) was undetectable. Among the beta 2 integrins, the levels of Mac-1 (CD11b) and p150.25(CD11c) were positive, while LFA-1 (CD11a) was not detectable. HLA class I and class II were also expressed. However, CR2 (CD21), CD57, CD44 and ICAM-1 (CD54) were not detected. Our study suggests that these techniques may aid in identifying changes in the expression levels of various surface antigens on conjunctival epithelial cells that could occur in ocular surface disease.

Dry eye and Meige's syndrome

AIMS To determine the relation between dry eye and Meige’s syndrome. METHODS 325 patients with dry eye were divided into those responsive to topical and other forms of treatment (n=276) and those who were not (n=49). A neuropsychiatric examination was performed to check for Meige’s syndrome in the latter group. RESULTS Twenty eight (57%) of the treatment unresponsive patients were diagnosed with Meige’s syndrome. CONCLUSIONS There is a subgroup of patients with dry eye who do not respond to simple therapy. More than half of these patients have Meige’s syndrome and need psychiatric, as well as ophthalmic, care.

Novel Noninvasive Sensitive Determination of Tear Volume Changes in Normal Cats

We describe a novel, high-resolution and noninvasive method for measuring tear volume changes in cats. The method entails photographing at the lid margin the tear meniscus area defined by instillation of 0.1% fluorescein solution into the cul-de-sac. The inferior tear meniscus area was obtained from the digitized images with computer-assisted software. The tear meniscus area increased in proportion to the saline volume applied into the conjunctival sac, which validates the technique. Furthermore, this technique detected with high sensitivity previously described increases in tear fluid secretion induced by the P2Y2 agonist. We demonstrate in cats that changes in conjunctival sac tear volume can be evaluated by measurement of its inferior tear meniscus area.

Lacrimation and salivation are not related to lymphocytic infiltration in lacrimal and salivary glands in MRL lpr/lpr mice

Sjogren’s syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into salivary and lacrimal glands leading to xerostomia and keratoconjunctivitis sicca.1–5 Lymphocytic infiltration into these glands has been demonstrated in several kinds of autoimmune mice, such as MRL/MP—+/+ (MRL/+), MRL/MP~lpr/lpr (MRL/lpr), NZB, NZB/W Fl hybrid, and NOD.6–8 Among these strains, MRL/lpr mice may be one of the best models for Sjogren’s syndrome, since marked infiltration of lymphocytes has been reported in lacrimal and salivary glands, which is indistinguishable from human disease.6 The infiltrated lymphocytes in the glands of MRL/lpr mice were largely CD4+ T-cells with lesser numbers of CD8+T-cells and B-cells,8–11 but CD4- CD8- double-negative T-cells were rarely detected.

Soluble interleukin-2 receptors and serum autoantibodies in dry eye patients: correlation with lacrimal gland function.

Purpose Sjögrens syndrome (SS) is a systemic autoimmune disease characterized by decreased lacrimal and salivary gland function causing dry eye and dry mouth. We examined several parameters as measures of lacrimal function and compared the serum soluble interleukin-2 receptor (sIL-2R) and serum autoantibodies as measures of systemic autoimmune condition. Methods Serum levels of sIL-2R, antinuclear antibody (ANA), and rheumatoid factor (RF), in 128 patients (16 men, 112 women; average age, 54.3 ± 12.5 years) were measured. The lacrimal function was evaluated clinically. Results There was no correlation between systemic condition and the cotton-thread test, Schirmer test with or without anesthesia, tear-clearance test, tear-function index, or the BUT. However, the Schirmer test with nasal stimulation and rose bengal and fluorescein staining was positively correlated with systemic parameters. Conclusion We show the correlation of serum sIL-2R, ANA, and RF to lacrimal function, which is good evidence that systemic immune conditions directly affect local lacrimal gland function.