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In homogenates of the human iris, the nonsteroidal anti-inflammatory drug (NSAID) S(+)flurbiprofen has been reported to inhibit cyclooxygenase-1 (COX-1) 70-fold more potently than in human whole blood. We hypothesized that this difference may be due to alternative splicing of COX-1 mRNA in the human iris or in whole blood. In this study, we have identified a similar COX-1 splice variant (COX-1SV) in both tissues with comparable COX-1/COX-1SV expression ratios. Therefore, we conclude that the difference in IC(50) values of S(+)flurbiprofen towards COX-1 in the human iris and human whole blood is not related to differences in the occurrence of spliced COX-1., (Copyright 2003 S. Karger AG, Basel)

Following the instillation of a drug into the eye, drainage mechanisms will commence at once. In this report, an attempt was made to assess the dynamics of an instilled nonsteroidal anti-inflammatory drug (NSAID), diflunisal, labeled with 1 MBq 99mTc followed by twenty minutes of scintigraphy. The results obtained with this labeled drug were compared with instillation of the same volume and activity of 99mTcO4-. Although the pertechnetate anion is an excellent and innocuous indicator for detecting the external lacrimal drainage system of the eye, it cannot visualize the internal structures. A clear scintigraphic difference was noted between labeled diflunisal and the pertechnetate anion. Scintigraphic activity surrounding the pupil of the eye provides evidence of visualization of the iris/ciliary body. This seems reasonable as the cyclooxygenase enzyme is located in this structure, and NSAIDs exert their mechanism of action via this complex. With this technology, visualization of some internal structures of the eye may be facilitated.

Aim: To evaluate the effects of beta blockers used in ophthalmology on the release of histamine from mixed cell preparations containing human leucocytes and basophils., Methods: A mixed leucocyte and basophil preparation was obtained from venous blood of healthy non-atopic volunteers. Cell preparations were then incubated with betaxolol, metipranolol, timolol, or carteolol. After incubation for 1 hour the histamine content of the supernatant was analysed by automated fluorometric analysis. Cell viability was tested by measuring lactate dehydrogenase (LDH) concentrations., Results: Betaxolol and metipranolol in concentrations between 10(-2) M and 10(-3) M liberated histamine from human blood cells in a dose dependent manner. Carteolol and timolol had no effect on histamine at these concentrations. At the same concentrations LDH was also detected in the supernatants of cell suspensions incubated with metipranolol or betaxolol., Conclusions: Betaxolol and metipranolol induce substantial histamine release from human leucocytes, probably as a result of their cytotoxic effect.

The purpose of the present study was to characterize the isoforms of cyclooxygenase (COX) in the human iris before and after stimulation with lipopolysaccharide (LPS) and to determine the selectivity of the nonsteroidal anti-inflammatory drug (NSAID), S(+) flurbiprofen, for inhibition of COX-1 and COX-2 in homogenates of this tissue. Spotblots were made of extracts of human iris in the absence and presence of LPS plus acetylsalicylic acid (aspirin). After reacting with anti-COX-1 and anti-COX-2 immunoglobulin G, the presence of both immunoreactive COX enzymes was substantiated using an indirect immunoperoxidase method. Authentic COX-1 and COX-2 were used as controls. Using an enzyme immune assay (EIA), the production of prostaglandin E2 (PGE2) was quantified in tissue homogenates of human iris under the same conditions as described above. S(+) flurbiprofen was added to tissue homogenates in order to determine the inhibitory effect on PGE2 production. Half maximal inhibitory concentrations (IC50) of S(+) flurbiprofen for the PGE2 production in the tissue homogenates were determined from concentration inhibition curves. The selectivity of S(+) flurbiprofen for inhibition of COX-1 was expressed as the ratio of IC50 for COX-2/COX-1. Spotblots of nonstimulated iris-extracts showed positive staining for COX-1 immunoreactivity (-ir) only. After incubation with LPS plus acetylsalicylic acid, positive staining was observed for both COX-1-ir and COX-2-ir. Concentrations of PGE2 released from homogenates of untreated iris varied from 1.5-4 ng/ml, and of LPS-stimulated tissue from 10-20 ng/ml of assay mixture. S(+) flurbiprofen inhibited PGE2 production of untreated tissue homogenates at an IC50 of 8 x 10(-10) M whereas, in the stimulated tissue, IC50 was found to be 3 x 10(-6) M. The selectivity of S(+) flurbiprofen for inhibition of constitutively present COX-1, relative to the inhibition of induced COX-2, was 3,600. Our results indicate that specific expression of COX isoforms in normal human iris was substantiated at the protein level by immunoreaction on spotblots. COX-1 represents the constitutively present enzyme, and COX-2 appears after stimulation with LPS. At the functional level, S(+) flurbiprofen possesses a specificity for COX-1 in inhibiting PGE2 production.

The purpose of this study was to assess the selectivity and potency of the nonsteroidal anti-inflammatory drug (NSAID), flurbiprofen, and its enantiomers in their inhibition of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). An assay was used with freshly drawn, heparinized human whole blood, incubated with 25 microM calcium ionophore A23187 during 60 min to produce thromboxane B2 (TXB2) by activity of COX-1 in platelets. Incubation with E. coli lipopolysaccharide (LPS) during 24 hr produced prostaglandin E2 (PGE2) by induction of COX-2 in monocytes, suppressing any possible contribution of COX-1 activity by the addition of acetylsalicylic acid. Concentration inhibition curves were determined with racemic, S(+), and R(-) flurbiprofen in final concentrations ranging from 10(-3) to 10(-10) M. The stereoselectivity of S(+) flurbiprofen vs. R(-) flurbiprofen, expressed as the reciprocal of the ratio of the concentrations giving 50% inhibition (IC50), is 340 for COX-1 and 56 for COX-2. The selectivity for COX-1 vs. COX-2, expressed as the reciprocal ratio of the IC50, was 32 for racemic, 16 for S(+), and 5.3 for R(-) flurbiprofen. Meloxicam in the same assay showed COX-2 selectivity with a ratio of 0.19.

Background: Several ocular side effects including uveitis, have been reported following topical beta blocker treatment for glaucoma and ocular hypertension. The incidence of these side effects was investigated in the Netherlands., Methods: A prospective observational design was used whereby monthly questionnaires were sent to all practising ophthalmologists in the Netherlands during 3 consecutive months. Questionnaires were returned at the end of each month. Any patient whose topical beta blocker therapy was altered because of an ocular reaction was noted on this questionnaire. Ophthalmologists who did not return their questionnaires were interviewed by telephone at the end of the study period. The number of patients using topical beta blockers was derived from drug sales figures., Results: 70% (328/467) of the ophthalmologists in the Netherlands participated in the study. During the 3 month study period 34 cases were reported: 15 patients had periorbital dermatitis, in eight patients eyelids and conjunctiva were affected, in seven patients the conjunctiva was affected, and four patients had punctate keratitis. The calculated incidence of ocular side effects during topical beta blocker therapy was 1.51 cases/1000 patient years., Conclusion: Topical beta blocker therapy is associated with few clinically important ocular side effects. No cases of uveitis were reported.

Purpose: To investigate whether mucosal immune responses directed against the ubiquitous parasite Toxoplasma gondii can be detected in tears of healthy humans., Methods: Nonstimulated tears and blood were obtained from 62 healthy humans (mean age, 35 +/- 10 [SD] years). Serum anti-T. gondii immunoglobulin titers were determined by Sabin-Feldman (SF) dye test. Western blot analysis was used to compare the anti-T. gondii repertoire in tears and serum, and antibody avidity was determined by urea elution. Diluted tear and serum samples were incubated with the intact parasite to determine whether the antibodies found in tears and serum are capable of binding to surface exposed antigens of T. gondii., Results: Eighty-one percent of the individuals tested had an anti-T. gondii IgA response in their tears, whereas only 23% had evidence of systemic immunity against the parasite. There was no apparent relation between chronic infection and presence of anti-T. gondii IgA in tears. Characteristically, the antigens recognized by the IgA antibodies in tears were often limited to at least one of four antigens with molecular weights of 74, 70, 49, and 34 kDa. The avidity of the anti-T. gondii IgA antibodies in tears was similar to the avidity of serum IgG antibodies. IgA antibodies directed against the 49- and 74-kDa antigens recognized epitopes exposed on the surface of the parasite., Conclusions: A major finding of this study is that tears of many individuals, chronically infected or not, contain IgA antibodies against T. gondii. It is not known whether these frequently observed antibody responses are the result of common mucosal immune responses against T. gondii or represent the natural antibody repertoire.

Aphakic cystoid macula edema, occurring after cataract extraction is ascribed to trauma-induced production of intra-ocular prostaglandins. Sufficient experimental and clinical evidence supports the use of prostaglandin synthesis inhibitors to countervail this clinical condition. The active S(+)-enantiomer of flurbiprofen, a prostaglandin synthesis inhibitor, has been formulated into a stereoselective, ballast free eyedrop solution in a concentration of 0.015%. Analysis by capillary zone electrophoresis shows shelf-life stability up to four years at room temperature of this enantiomer. The inhibitory effect on the synthesis of prostaglandins as measured on a homogenate bovine iris/ciliary body, remained unaffected during a shelf-life period of three years.

A presence of cystatins, inhibitors of cysteine protease, was investigated in tears of patients with different corneal pathologies. Tear fluid samples were collected with glass capillaries in 28 patients (28 eyes) and 15 healthy controls (15 eyes). Only after corneal transplantation (8) or in corneal dystrophy (6) but not after cataract extraction (5) or other traumatic conditions of the cornea (9) was a significant difference in inhibitory activity of cystatins measured in comparison with the 15 controls. In this study it could not be decided whether the lower level of cystatins was related to the cause or the effect of the condition of the eye.

One of the inflammatory responses of the eye to local application of platelet-activating factor (PAF) is oedema of the conjunctiva, caused by extravasation of plasma. Aim of the study was to investigate if fluorescein would leak from the blood into the tears together with plasma protein after application of PAF to the eye. Fluorescein was given intraperitoneally 30 min prior to application of 25 microl of 0.1% solution of PAF. Thirty min after PAF the tear film was collected by washing the surface of the eye with 25 microl of phosphate buffered saline (PBS). Fluorescein in eye washings and in plasma was measured by fluorophotometry and albumin by immunodiffusion. Both fluorescein and albumin appeared in a related fashion in tears, being absent in washings of placebo-treated control eyes. Extravasation of fluorescein can be used as a measure for plasma leakage in the conjunctiva with the advantage over the Evans Blue method that the former is a non-invasive method.

Both nitric oxide and prostaglandins induce vasodilatation which is an important feature of local inflammation. The purpose of the study described here was to investigate a possible interaction between these two types of mediators in an experimental model of allergic conjunctivitis. A conjunctival allergic reaction was induced with antigen in sensitized guinea pigs. Conjunctival vascular permeability changes were evaluated with the prophylactic use of an inhibitor of nitric oxide synthase (L-NAME) and a cycloxygenase inhibitor (indomethacin). To study a possible interaction between nitric oxide and prostaglandin synthesis in the acute phase of allergic conjunctivitis, the levels of nitrite and PGE2 were determined in lavage fluid. The prophylactic use of L-NAME on the formation of conjunctival edema in response to topical PGD2 administration was studied by measurement of albumin levels in lavage fluid. Both nitric oxide and PGE2 are synthesized in response to antigen provocation and after histamine administration. Nitric oxide and PGE2 are produced simultaneously in the conjunctiva and they showed identical synthesis profiles in response to antigen provocation. Pretreatment with L-NAME inhibited the synthesis of PGE2 whereas exogenous administration of nitric oxide increased the level of PGE2 in lavage fluid. Prophylactic treatment with L-NAME significantly inhibited the PGD2 induced albumin extravasation. Nitric oxide seems to play an important role in the acute phase of allergic conjunctivitis it may stimulate PGE2 production and acts as a secondary mediator in PGD2 and histamine induced conjunctival edema.

In a previous paper we reported the presence of components in human tear fluid that block the interaction of proteins with plastic surfaces, interfering with tear protein ELISA and proposed the term coating inhibiting activity. The purpose of the study presented here was to further analyse these components. Coating inhibitory activity in human reflex tears was analysed by lectin affinity chromatography, using the agarose bound lectin Artocarpus integrifolia agglutinin (Jacalin), gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting and Jacalin staining. For coating inhibitory activity assay in experimental tear samples, the binding of the protein Avidin-conjugated horseradish peroxidase to the polystyrene surface of ELISA micro-titer plate wells, preincubated with the experimental tear samples was measured. In addition, tears were incubated with scrapings of the ELISA plates used in the assay and with six different types of contact lenses (two rigid gas permeable and four hydrogel soft contact lenses) for analysis of adsorbed components. Lectin affinity chromatography of tears yielded a Jacalin-binding and a non-Jacalin-binding preparation, both exhibiting coating inhibitory activity but representing chemically different preparations as observed by SDS-PAGE. After performing gel filtration, coating inhibitory activity eluted with similar retention in both preparations. In fractions exhibiting activity, tear proteins of low molecular weight (< 40 kDa) were detected. Among these, two Jacalin-binding glycoproteins were detected; a major component of approximately 28 kDa and a somewhat smaller minor component. All low molecular weight components were also detected on the scrapings, incubated with tears. The possibility that coating inhibitory activity in tears might reside in a component of larger molecular size can however not be excluded. The human tear proteins secretory Immunoglobulin A, lactoferrin and lysozyme are not involved in coating inhibition. On one of the two rigid gas permeable contact lenses incubated with the tears, the 28 kDa glycoprotein was detected. From the data obtained in our study we conclude that coating inhibitory activity in tears seems to be associated with multiple components of low molecular weight.

The role of nitric oxide in allergic conjunctivitis was studied in a guinea pig model. The eyes of sensitized guinea pigs were challenged with ovalbumin (20 micrograms per eye) or histamine (20 micrograms per eye). Synthesis of nitric oxide (NO) was inhibited using L-NAME (200 micrograms per eye) or aminoguanidine (200 micrograms per eye). The formation of conjunctival edema was graded and levels of nitrite, a breakdown product of nitric oxide were measured in lavage fluid. Conjunctival vasopermeability was determined by measuring the albumin concentration in the fluid on the surface of the eye (lavage fluid). Animals were treated with sodium nitroprusside (SNP) or phenylephrine after which histamine induced conjunctival vasopermeability changes were measured. Drugs were administered topically with the other eye serving as a control. Both ovalbumin and histamine produced a marked inflammatory response including hyperaemia and edema. At the top of the inflammatory response occurring 30 min after challenge, increased levels of nitrite, a breakdown product of NO, were measured in lavage fluid. Prophylactic treatment with L-NAME or aminoguanidine resulted in a significant inhibition of the NO synthesis. Both L-NAME and aminoguanidine decreased conjunctival vascular permeability and edema formation significantly. Administration of SNP resulted in a marked dilatation of conjunctival blood vessels and produced a dose-dependent increase of vascular permeability. Addition of SNP to histamine significantly enhanced conjunctival edema and potentiated vascular permeability. These results indicate that NO is produced in the acute phase of allergic conjunctivitis and mediates vasodilatation after topical provocation with ovalbumin or histamine in sensitized guinea pigs. The resulting increase of the conjunctival blood flow subsequently increases the vascular permeability and enhances conjunctival edema formation. Inhibition of NO synthesis leads to a reduction of conjunctival hyperaemia and subsequently reduces the formation of edema.

The role of nitric oxide (NO) as an inflammatory mediator in the mechanism of increased microvascular permeability was examined in a guinea pig model of allergic conjunctivitis. Topical challenge with antigen, compound 48/80, histamine or platelet activating factor (PAF) resulted in a marked increase of the conjunctival vascular permeability. Vascular permeability was determined by measuring the albumin content in the lavage fluid of the challenged eyes after 30 min. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) eyedrops caused a significant inhibition of the clinical score and the vascular permeability after challenge with either antigen, histamine or PAF. Aminoguanidine prophylaxis also resulted in a significant inhibition of both the clinical score and vascular permeability in response to all the used provocative agents except PAF. Our observations indicate that NO is an important factor in the induction of the vascular permeability provoked by histamine but seems to play no role in the mechanism by which PAF exerts increased vascular permeability in the conjunctiva.

Purpose: The presence of corneal opacities associated with dacryoadenitis and lacrimal gland destruction has led investigators to consider MRL/Mp mice as models for band keratopathy and Sjögren syndrome. In this study, the authors examined the time course of the corneal opacification and investigated whether the opacities were associated with altered serum levels of parathyroid hormone, calcium, and phosphorus, as well as quantitative and qualitative differences in tear production., Methods: Corneas were analyzed microscopically and tear fluid production was measured by a modified Schirmer test., Results: Corneal lesions were observed as early as the fifth week after birth. The lesions consisted of calcium phosphate and appeared as punctate, crystalline opacities located subeithelially. Lesions were present in 72% (56 of 78) of the MRL/Mp mice, with no significant difference in incidence between MRL/Mp +/+ and MRL/Mp lpr/lpr mice. Corneal calcification was occasionally associated with a self-limiting keratitis and neovascularization. In control mice, corneal opacities were not observed before the animals were 6 months of age. Levels of circulating parathyroid hormone decreased significantly during the first 16 weeks of age in MRL/Mp mice. In addition, MRL/Mp mice of both sexes had a significantly lower tear fluid production as compared to BALB/c mice of the same age., Conclusion: Because corneal lesions start to develop in 5-week-old MRL/Mp mice, thereby preceding the clinical signs of systemic autoimmune disease, and may develop in 6-month-old nonautoimmune-prone mice, it is suggested that calcification develops independent of the systemic autoimmune disease and might be restricted to the cornea.

Secretory immunoglobulin A, lactoferrin, lysozyme and tear specific pre-albumin were analyzed in stimulated tear fluid of 25 diabetic patients without retinopathy and in 29 diabetic patients with (pre) proliferative retinopathy using high performance liquid chromatography. Results were compared to those obtained in 26 healthy controls to determine the effect of diabetes mellitus on the exocrine function of the main lacrimal gland. Sodium dodecyl sulfate polyacrylamide gel electrophoresis onto minigels was performed on 20 tear samples for verification of high performance liquid chromatography fractions recorded. The mean total protein values in tear fluid (Bradford assay) of diabetics without retinopathy, with retinopathy and healthy controls did not differ significantly (mean in mg/ml +/- SD: 6.4 +/- 2.2, 5.9 +/- 2.0 and 5.7 +/- 1.7, respectively; Mann-Whitney; p > 0.02). High performance liquid chromatography showed an increased secretory immunoglobulin A and decreased peak 5 OD280 (+56% and -38%, respectively; p < 0.02) in patients without retinopathy, whereas in patients with retinopathy lysozyme was increased (+27%; p < 0.01) and tear specific pre-albumin and peak 5 OD280 decreased (-24% and -42%, respectively; p < 0.04), when compared to healthy controls. These inconsistent differences do not uniformly suggest an exocrine dysfunction of the main lacrimal gland in diabetic patients.

In experimental immunogenic keratitis, provoked in rabbits by intracorneal injection of 20 microliters of human serum albumin (HSA), various anti-inflammatory agents were studied in their effects on corneal edema, neovascularisation and leukocyte infiltration. Prophylactic treatment with a corticosteroid completely prevented the occurrence of keratitis. Nonsteroidal anti-inflammatory drugs such as a cyclooxygenase inhibitor partly prevented neovascularisation and corneal edema, a lipoxygenase inhibitor, a leukotriene antagonist or platelet-activating factor (PAF)-antagonist BN 52021 partially prevented mainly leukocyte infiltration. Prophylactic topical treatment with the poly-unsaturated fatty acids eicosapentaenoic acid and columbinic acid or a dietary supplement with fish oil showed less symptoms of keratitis in all respects.

The inhibitors of prostaglandin (PG) or leukotriene (LT) synthesis and antagonists of platelet-activating factor (PAF) or LTs are inhibitory in experimental keratitis and clinical symptoms of keratitis are reproduced by application of these lipid mediators. This suggests that PGE2, LTB4, LTD4, and PAF are involved in experimental immunogenic and toxic keratitis. The objective of the present study is the measurement of the concentrations of lipid mediators in the aqueous humour and their release by the cornea and iris during keratitis. In both inflammatory models the concentrations of PGE2, LTB4, LTD4, and PAF in the aqueous humour were significantly increased as compared to their controls. The release of PGE2, LTB4 and LTD4 from the cornea, and of PGE2, LTB4, and PAF from the iris was significantly increased compared to that from control tissues. The results are consistent with a role for these lipid mediators in the inflammatory models. Combined therapeutic use of synthesis inhibitors or antagonists of these mediators in eye inflammation seems possible and may serve as an alternative to topical corticosteroid therapy.

Previously we have reported that various pathologic corneas exhibited a "diseased" two-band corneal aldehyde dehydrogenase (ALDH) zymogram after native polyacrylamide gel electrophoresis as compared with the three bands in the normal human cornea. Experimentally, such a "diseased" zymogram pattern could be induced by addition of reduced glutathione (GSH) to the normal corneal epithelial extract. This finding suggests that in vivo the conformation of corneal ALDH may be related to changes in the GSH redox system during the process of corneal diseases. To investigate this hypothesis in keratoconus corneal epithelial extracts and a separate group comprising other corneal disorders, mainly herpes keratitis, we indirectly measured the GSH turnover by assaying the activity of glutathione reductase (GR) which is responsible in producing GSH and glutathione s-transferase (GST), which converts GSH into mercapturic acid. Our results indicate that there is a correlation between the activity of GR and GST in the normal and the separate group of corneal disorders. Because GST is the first enzyme in the mercapturic acid pathway, which detoxifies xenobiotic substrates including aldehydes, as by-products of membrane lipid peroxidation, an elevated GSH turnover might be necessary to counteract oxidative threats. However, no correlation was found between corneal ALDH level with either GR or GST. On the other hand, keratoconus samples demonstrated a distinct enzymatic behavior that was in concordance with our earlier result in the corneal ALDH zymogram after isoelectric focusing. Furthermore, analysis of our several studies tends to support the proposed structural function of ALDH in human cornea.

Using a modified turbidimetric assay to determine the protein concentration in human tears by precipitation with trichloroacetic acid (TCA) we found lower protein concentrations if compared with other methods for protein determination. This implies that a factor in human tears is able to inhibit the precipitation of protein by TCA. Earlier a coating inhibitory factor in human tears was described which is able to prevent coating of a polyacrylate surface by proteins using a ELISA methodology. Because of the similarity in its behaviour towards protein we investigated whether the same factor could be responsible for both inhibitory effects. A pool of human tears was separated into various fractions using HPLC whereafter inhibitory activity in the turbidimetric and the coating assay could be found in the same fractions. Characterization of the inhibitory factor was performed by minigel-electrophoresis (SDS-PAGE), after which blotting and staining with a lectin (Jacalin) revealed two subunits of a glycoprotein with a molecular weight of 30 and 70 kD. The inhibitory factor also could be isolated if human tears were incubated for 30 min at 100 degrees C whereafter precipitated protein was removed by centrifugation. Inhibitory activity could be detected in the supernatant and an identical glycoprotein profile could be produced after staining with lectin (Jacalin). The results of this study suggest that a soluble glycoprotein serves as a coating and precipitation inhibitor in tears and may play an important role in the protein to protein interaction on the surface of the eye.

Tear fluid analysis was performed in 44 patients with primary SS, 21 patients suspected of having primary SS in whom the syndrome had been excluded, and 24 control subjects. In the primary SS patients the tear fluid levels of lactoferrin and a1-antitrypsin were increased and the tear fluid levels peroxidase, lysozyme and amylase were decreased. However, a considerable overlap in the concentrations of all of the tested substances in the different groups was found and the measurement of these substances cannot be advocated for diagnostic use.

In this study we investigated the properties of corneal aldehyde dehydrogenase (ALDH) in keratoconus corneas using various electrophoretic techniques combined with immunochemical and zymographic identification. Normal corneas and other pathologic corneal buttons obtained during keratoplastic surgery were used as a control. A significant (p < 0.001) lower enzymatic activity was found in keratoconus epithelial extracts (3.1 +/- 2.1 IU/mg protein) compared with normal controls (5.5 +/- 2.6 IU/mg protein), whereas no significant differences were observed in the stromal and endothelial extracts. No significant differences were observed for the corneal ALDH thermolability behavior, nor did any marked changes occur in the position of the 54- and 88-kDa species when comparing the pathologic corneas and normal controls. On the other hand, isoelectric focusing analysis showed a different pattern for the pathologic corneas as compared with controls. Moreover, native-polyacrylamide gel electrophoresis analysis showed that normal corneas exhibit three bands, whereas keratoconus and other pathologic corneas only show two bands. The shift from the three-band pattern to the two-band pattern could be reproduced in vitro using reducing agents, such as glutathione.

Topical application of 250 micrograms of platelet activating factor (PAF) or 5 micrograms of prostaglandin E2 (PGE2) to the rabbit's eye is followed by a rise in intraocular pressure (IOP). The occurrence of PAF in the anterior chamber is held responsible for this effect, as appears from the observed concentrations of PAF in a ng/ml-range in the aqueous humor and from the inhibition in the rise of IOP by systemic pretreatment of rabbits with the PAF-antagonist BN 52021 (10 mg/kg). Corneal and conjunctival edema, occurring after application of PAF, are not influenced by BN 52021. Thus, these effects may be ascribed to the non-specific membrane pertubant action of the high local concentration of PAF, necessary to achieve effective concentrations of PAF in the anterior chamber.

The major soluble protein in the bovine cornea (BCP 54) has recently been identified as a class 3 aldehyde dehydrogenase. An enzymatic and even a possible structural role of this protein in the mammalian cornea has been proposed. Earlier we showed that the human cornea contains the same enzyme but with a different substrate specificity, compared to the bovine. Moreover, the enzymatic activity was harbored in the dimeric 88-kD species. Genetic variants have been found for several mammalian ocular aldehyde dehydrogenases. In this study we investigated whether such variants were also present in the human cornea by using a zymography technique for aldehyde dehydrogenase activity and immunoblotting with rabbit anti-BCP 54 and lectin staining after isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We investigated 50 individual human corneal epithelial extracts and found one common IEF variant (n = 47) and two "rare" IEF variants in three individuals. Analysis of these patterns indicates that the observed IEF profiles may be caused by posttranslational events.

Ophthalmological examination of patients with primary Sjögren's syndrome (SS) (n = 44) taken randomly from a rheumatological practice revealed that in this population group the eyes were frequently but not seriously involved (n = 42). Primary SS patients (n = 44) could not be separated from patients suspected of primary SS, in whom the diagnosis had been excluded (n = 21) on the basis of ocular examination, but they were clearly distinguishable from healthy controls (n = 26). The diagnostic value of the tests currently in use to establish the diagnosis keratoconjunctivitis sicca in primary SS such as Schirmer's, tear film break up time and rose bengal staining tests appeared to be limited. These results demonstrate the heterogeneity of the syndrome, the influence of patient selection on patient characteristics in different primary SS populations and the limited value of using the present classification criteria for the diagnosis primary SS in individual patients.

Aldehyde dehydrogenase has recently been shown to be one of the major soluble proteins in the mammalian cornea. The enzyme has a subunit molecular weight of 54 kd and gel filtration experiments indicate that a dimer molecule is the enzymatically active species. The purpose of the studies described here was to investigate whether oligomeric forms of this enzyme could also be detected using the much faster SDS-PAGE mini-gel electrophoresis technique combined with immunoblotting and "in gel" enzyme detection. Low temperature treatment of samples prior to electrophoresis revealed that both human and bovine corneal ALDH are mainly present as a 54 kd and as a dimer molecule with an apparent molecular weight of 88 kd. Bovine corneal ALDH also contained larger oligomers with a molecular weight of 110, 154 and 210 kd respectively. The classical 3 minutes boiling procedure prior to SDS-PAGE dissociated the oligomers into the 54 kd subunit. Zymography experiments showed that enzyme activity was only present in the 88 kd form of corneal ALDH. Pretreatment of corneal ALDH at various temperatures showed that the temperature induced shift of the 88 kd species to the 54 kd subunit paralleled the decrease in enzymatic activity. The fact that reduction of samples with DTT did not dissociate the 88 kd form suggests that disulfide bridge formation is not involved in the oligomerisation of corneal ALDH.

The influence of viscolysers on the precorneal residence of a fluorescent tracer was determined, using slit lamp fluorophotometry. The solution acceptability was evaluated by the volunteers by answering a standard questionnaire. The relationship between precorneal retention of viscous eye drops, discomfort and tear fluid composition after instillation of various cellulosic solutions was examined. Irritating hydroxypropylcellulose solution increases the total protein concentration of tears, without change in the ratio of lysozyme to total protein.

The identity of glycoproteins in stimulated normal human tears was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of tears onto minigels, blotting, and subsequent incubation with different biotinylated lectins (concanavalin A [Con A], peanut agglutinin [PNA], glycine max agglutinin [SBA], Phaseolus vulgaris agglutinin, wheat germ agglutinin [WGA, native form], Artocarpus integrifolia agglutinin [Jacalin], and Pisum sativum agglutinin). Control proteins included purified secretory immunoglobulin A (sIgA) from human colostrum, human milk lactoferrin, and chicken-egg lysozyme. All samples were prepared in a denaturing (SDS) buffer under nonreducing and reducing conditions. The sIgA in tears and IgA (alpha) heavy chain fragments (reduced sample) were identified with most of the lectins tested. A particular high molecular weight (greater than 200 kD) protein fraction in tears that just entered the separation gel on SDS-PAGE was detected with WGA and Jacalin. This fraction stain poorly with silver. Tear lactoferrin was identified with all lectins used, although binding was low with SBA. Purified milk lactoferrin showed a poor reaction with Jacalin, but a protein in tears of similar mobility bound this lectin (nonreduced samples). Under both nonreducing and reducing conditions, tear-specific prealbumin in tears did not bind any of the lectins tested. Tear lysozyme only reacted with lectin after reduction. The techniques described may provide additional valuable information in addition to commonly used methods for tear protein analysis and further knowledge concerning the role of glycoproteins on the ocular surface.

The major soluble protein in bovine corneal epithelial extracts is a 54 kD protein (BCP 54) which has recently been identified as the corneal aldehyde dehydrogenase. Although ALDH activity has been reported in human corneal extracts it was not yet clear whether this was identical with the 54 kD protein described in bovine corneas. To investigate this question, we studied human corneal extracts for the presence of ALDH using enzyme analysis, SDS-PAGE, native electrophoresis, isoelectric focusing and immunoblotting techniques. The corneal epithelium was the most active layer (8.46 +/- 1.9 IU/mg protein) followed by the stroma (2.83 +/- 0.56 IU/mg protein) and endothelium (0.06-3.6 IU/mg protein). When comparing substrate specificity between human and bovine corneal ALDH, using NADP as coenzyme, it was shown that the human enzyme preferred benzaldehyde whereas the bovine enzyme revealed the strongest enzymatic activity with hexanal. Human corneal ALDH was partly inhibited by disulfiram. Bovine and human cornea ALDH lost their enzymatic activity after heating at temperatures above 56 degrees C. Both human and bovine corneal extracts contained a prominent 54 kD protein which reacted with a rabbit anti BCP 54 antibody. Isoelectric focusing followed by enzyme staining in the gel revealed 5 human corneal isozyme species and 4 in bovine corneal extracts, migrating at a pH between 6.5 and 7.0. All isozymes could also be detected after immunoblotting with a rabbit anti BCP 54 antibody.

An automated Minigel electrophoresis system (PhastSystem, Pharmacia, Uppsala, Sweden) was tested for human tear protein analysis. Tear samples were treated under nonreducing or reducing conditions before sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE). Micro-amounts of tears (2 microliters) were sufficient for analysis and separation and visualization of proteins were completed within 2 hr. Tear proteins were identified using purified control proteins and immunoblotting techniques, using antisera against immunoglobulin (Ig) A (alpha) heavy chains, Ig heavy and light chains, secretory components and lactoferrin. In nonreduced tears, lactoferrin (seen as a double band), serum albumin, tear-specific prealbumin (TSPA), and lysozyme were clearly separated. Secretory IgA (sIgA) was seen as a smear on top of the gel. Immunostaining also showed a major Ig light chain containing protein. After reduction, the protein profiles showed marked changes. In reduced tears, immunoglobulin heavy and light chains (molecular weight [MW]: 64 and 28 kD, respectively) were detected on the SDS-PAGE profile after immunostaining, and represented disulfide cleavage fragments, which originated from sIgA. Reduction resulted in the liberation of the secretory component piece (MW: 85 kD), which was found to co-migrate with the tear lactoferrin bands. Both lactoferrin and serum albumin acted as larger proteins on SDS-PAGE after reduction. The authors found that the two methods of sample treatment, before electrophoresis, resulted in marked differences on the electropherograms.

Antagonists of leukotriene D4 (LTD4), 3-[7-(2-n-propyl-3-hydroxy-4-acetyl- phenoxy)-propoxy-2-methyl-3,4-dihydro-4-8-n-propyl-2H-1-benzopyran- 2-yl]-propionic acid (SC-39070) and 7-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-2-hydroxy- propoxy]-4-methyl- 2-oxo-2H-1-benzopyran-3-acetic acid (FPL-60319) were locally administered to eyes of rabbits with immunogenic keratitis. The period of circular leukocyte infiltration (Wessely's ring) in the cornea was markedly decreased. Edema and neovascularization of the cornea were not influenced.

Rabbit eyes were enucleated and frozen in liquid nitrogen. The vitreous was removed and analyzed for prostaglandin E2 (PGE2). The mean level (+/- SEM) of PGE2 in the anterior as well as in the posterior vitreous was 0.09 +/- 0.016 ng/ml (n = 12 rabbits). Animals pretreated with indomethacin 15 min before death, in order to prevent the formation of prostaglandin during enucleation, showed in both the anterior and posterior vitreous mean PGE2 levels of 0.04 +/- 0.008 ng/ml (n = 12) which were significantly lower. Ocular trauma such as paracentesis of the anterior chamber, indentation of the sclera or laser photocoagulation in the fundus 1 h before death of the animals did not increase the concentration of PGE2 in the vitreous humor. There is no evidence either for local release of PGE2 from tissues in the fundus of the eye or for diffusion of PGE2 from the anterior chamber into the vitreous humor.

Tears from normal (n = 5) and serum IgA deficient (n = 3) individuals were investigated for the presence of secretory Immunoglobulin A (sIgA), sIgM and free secretory component (SC) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using 10-15% gradient minigels (PhastSystem), followed by immunoblotting using various immunological probes. Tear samples were treated in denaturing (SDS) sample buffer under non-reducing as well as reducing conditions, prior to analysis. All normal tear samples contained sIgA as well as free SC (estimated MW: 82kD) but only traces of IgM. Tears from the three serum IgA deficient subjects lacked sIgA but did contain free SC. In two of them sIgM was clearly detected and after treatment of tears with reducing agent, IgM (mu) heavy chain fragments (estimated MW: 78kD) were identified and could be distinguished from other tear proteins after SDS-PAGE. These findings indicate lacrimal secretion of free secretory component, even in the absence of its ligand. On the ocular surface, sIgM may play a compensatory role in IgA deficiency.

Fish lipids contain large amounts of eicosapentaenoic acid (EPA) and docosahexaenoic acid. These fatty acids are known to have an influence on prostaglandin (PG) and leukotriene (LT) synthesis. We studied the effect of a fish oil dietary supplement on an immune-complex-induced keratitis of the rabbit eye and compared it with the effect of a sunflower seed oil dietary supplement, rich in linoleic acid. Immune complex keratitis induced by intrastromal injection of human serum albumin (HSA) was characterized by leukocyte infiltrate, neovascularization, and corneal edema. Animals given a fish oil diet showed significantly less leukocyte infiltrate, neovascularization, and corneal edema, compared to those given a sunflower seed oil diet.

The anti-inflammatory compound d-ibuprofen has been investigated for anti-inflammatory and cyclooxygenase inhibitory activity in ocular inflammation induced by paracentesis of eyes of living rabbits. d-Ibuprofen is the dextro-rotary isomer of ibuprofen, a potent non-steroidal anti-inflammatory agent. Eyes pretreated topically with d-ibuprofen 0.8% showed a significant inhibition of aqueous protein (73.0%) and prostaglandin E2 (96.4%) increase after paracentesis as compared to paracentesized untreated fellow eyes and control eyes. In aqueous humor no significant correlation between the increase in prostaglandin E2 and protein could be established after paracentesis. These results indicate that d-ibuprofen could be a useful ocular anti-inflammatory agent as cyclooxygenase inhibitor.

A simple assay for the determination of sialic acid (N-acetylneuraminic acid) in human tear fluid was evaluated. Sialic acid, terminally bound on carbohydrate side-chains of glycoproteins, was released after treatment with neuraminidase and measured by an enzymatic colorimetric test. Tear fluid samples were collected from ten healthy adults, using glass capillaries and cellulose sponges. Sialic acid levels in tears collected with sponges (0.8-1.8 mmol l-1) did not differ significantly from those found in capillary tears (0.9-1.8 mmol l-1). Sialic acid, expressed as mmol g-1 protein, was significantly lower in tears collected with sponges (0.18-0.32 mmol g-1) than with capillaries (0.19-0.42 mmol g-1). Recovery of sialic acid and protein after incubation of cellulose sponges with tears was more than 99%. Sialic acid levels in human tears, which had been centrifuged to remove insoluble material, remained unchanged. Furthermore, tear sialic acid activity did not pass a filter with a molecular weight cut-off point of 10,000. Our data indicate that with the assay used in this report, sialic acid in tears is not due to secretory immunoglobulin A (sIgA), lactoferrin and lysozyme. The fact that the major tear proteins do not contribute to the sialic acid levels detected in tears suggests that other as yet unknown soluble glycoproteins are involved.

The protective effect exerted by BN 52021 a specific PAF-receptor antagonist in experimentally induced ocular inflammatory disorders led us to investigate the possible presence of specific receptors for PAF in rabbit iris and ciliary body. Two classes of PAF binding sites were found in isolated iris and ciliary process of pigmented rabbit eyes: a high affinity site Kd1 congruent to 4.9 +/- 0.47 nM, Bmax1 congruent to 3.17 +/- 0.50 pmoles/mg protein, a low affinity sites Kd2 congruent to 11.6 +/- 0.33 nM, Bmax2 congruent to 12.46 +/- 2.3 pmoles/mg protein for iris. The specific binding was not affected by lyso-PAF the biologically inactive precursor and metabolite of PAF, up to 10(-6) M; inhibition by unlabelled PAF demonstrated a biphasic curve partially antagonized by BN 52021. The present results demonstrate the presence of specific binding sites for PAF in rabbit eyes which could mediate the action of this mediator in eye inflammatory processes and explain the protective effect observed with BN 52021.

Human tear fluid has plasminogen activator activity. The type of plasminogen activator activity in unstimulated and stimulated tears was determined, using antibodies that specifically neutralize tissue plasminogen activator or urokinase. All plasminogen activator activity was tissue plasminogen activator-related in both types of tears. Correlations between activities of beta-hexosaminidase, lysozyme and lactate dehydrogenase with tissue plasminogen activator activity indicate that the contribution to plasminogen activator activity of conjunctival and corneal epithelium is more important in unstimulated tears than stimulated tears. In stimulated tears the tissue plasminogen activator activity originates mainly from the lacrimal gland. It is suggested that a constant concentration of plasminogen activator is released from the lacrimal gland and that this concentration is independent of the secretion rate of tear fluid and that the release from the conjunctiva is due to desquamation of cells.

A variety of enzymes were identified in rat tears and lacrimal gland fluid. The use of a tapered glass cannula in the opening of the excretory duct was found to be an useful method to collect samples of rat lacrimal gland fluid, i.e., the fluid directly originated from the main excretory duct of the lacrimal gland, uncontaminated by secretions from the Harderian gland and from desquamating conjunctival and corneal epithelial cells. Based upon comparison of the enzyme pattern in tears, lacrimal gland fluid and the lacrimal gland tissue, we concluded that the lacrimal gland is the primary tissue source for peroxidase (POD), amylase (AMY) and total protein in rat tears, while plasminogen activator and lactatedehydrogenase (LDH) may be present in tears primarily as secretion products from other ocular tissue sources. beta-hexosaminidase (beta-hex) is released from the lacrimal gland and from other ocular tissue sources as well.

The existence of specific binding sites by using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF) was investigated on iris and ciliary body tissue of pigmented rabbit eyes. The binding was saturable, specific, time-dependent and reversible. Scatchard's analysis indicated the presence of two types of binding sites with a Kd1 4.90 +/- 0.47 nM, a Kd2 11.60 +/- 0.33 nM, a Bmax1 3.17 +/- 0.50 pmol/mg protein and a Bmax2 12.45 +/- 2.30 pmol/mg protein for iris tissue, and a Kd1 5.71 +/- 0.09 nM, a K2d 24.40 +/- 0.91 nM, a Bmax1 3.41 +/- 1.00 pmol/mg protein and a Bmax2 16.60 +/- 0.51 pmol/mg protein for ciliary body. The binding was fully displaced by unlabelled PAF in both iris and ciliary body preparations, and partially inhibited by the PAF antagonist BN 52021 in iris tissue.

As part of a combined ophthalmological, genetic, clinical, biochemical, ultrastructural and electro-physiological study of albinism we have examined over one hundred albinos, together with their heterozygote family members. Given this substantial number, we have developed a diagnostic protocol to facilitate albino classification and heterozygote detection. The major difficulty in the detection and differential diagnosis of albinism is that for a given albino, not all symptoms, either ophthalmological or cutaneous, may be manifest, whereas several may be in a non-albino. To compensate for the wide diversity in albino expression, diagnosis is typically based on the results of combinations of tests.

In rabbits inflammation of the cornea was induced by intrastromal injection of horse serum. Between 2 and 4 weeks after injection, infiltration of the cornea with leukocytes and neovascularization could be observed. During this period, the rabbits were killed and their corneas analyzed for protein, alkaline phosphatase, acid phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase. The enzyme activities in the inflamed corneal stroma reflect the high lysosomal activity, which probably originates from the leukocytes. The enzyme activities in the epithelium indicate that the tissue is abnormal and undergoing repair processes.

Intrastromal injection with human serum albumin (HSA) in the rabbit cornea induced edema and a ring-shaped leukocyte infiltrate followed by neovascularization. The effect of topically administered lipoxygenase and cyclooxygenase inhibitors on this inflammatory keratitis was studied. The lipoxygenase inhibitors Bay 08276 and Rev 5901 and the cyclooxygenase inhibitor suprofen were given as 1% eye drops three times daily during the experiment. In eyes treated with lipoxygenase inhibitors leukocyte infiltration, neovascularization and edema formation decreased. In eyes treated with a cyclooxygenase inhibitor the period of neovascularization was slightly shortened and corneal edema decreased. No influence on leukocyte infiltration was seen.