Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.

Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.

[Morphological and histochemical studies on the development of human conjunctival goblet cells].

This report deals with the development of human conjunctival goblet cells. Fifty-six eyes of human embryos and fetus ranging from 5 to 41 weeks of gestational age were used in this study. Glycosaminoglycans in the goblet cells were investigated histochemically using 1% alcian blue staining (pH = 2.5) and PAS reagent staining and sialidase (neuraminidase) digestion. At 8 weeks, goblet cells appeared in the forniceal area, and they extended to the palpebral and bulbar conjunctiva. These cells were already similar to adult goblet cells. Alcian blue staining and the enzyme digestion method revealed the existence of sialic acid in the goblet cells from 9 weeks. These results suggest that at the early developmental stage the goblet cells develop from the forniceal area. It is also indicated that the goblet cells in the early developmental stage are already similar to cells in the adult stage because they contain mainly sialic acid as glycosaminoglycans.

Beta adrenergic binding sites in the human eye: an autoradiographic study.

Beta adrenergic binding sites were localized and characterized in the human eye by means of "in vitro" autoradiography, using [125I] (-) iodocyanopindolol (125ICYP) as radioligand. Binding sites were visualized by apposition of isotope sensitive film to slide mounted eye sections. Receptor sites were present in the extraocular muscles, in the conjunctiva, in the epithelium and endothelium of the cornea, in the trabeculum and in the ciliary muscle. They were also present in the lens epithelium and in the retina. The pigmented ocular structures were heavily labelled but the binding was nonspecific. Characterization of these binding sites was achieved by testing the ability of selective adrenergic compounds to displace 125ICYP binding. These studies suggested that the majority of adrenergic binding sites in nonpigmented structures of human eye were of a beta2 type.

Neoglycoprotein binding distinguishes distinct zones in the epithelia of the porcine eye.

The presence and location of specific endogenous lectins in the anterior epithelia of the porcine eye were determined by histochemical staining using biotinylated glycosylated carrier molecules. Endogenous lectins with specificities for galactosides, alpha-mannosides and beta-xylosides were present in the conjunctival epithelium, but the corneal anterior epithelium appeared essentially free of histochemically demonstrable endogenous lectins. The corneal posterior epithelium contained a rich and complex set of endogenous lectins. The transitional area between conjunctival and corneal anterior epithelia at the limbus cornea contained, in striking contrast to either of the epithelia bordering it, cells expressing endogenous lectins in considerable wealth and variety.

Conjunctival cell glycoprotein pattern of healthy persons and of patients with primary Sjögren's syndrome--light microscopical investigation using lectin probes.

Formalin-fixed biopsies of conjunctival tissue from the lower fornix of 10 healthy persons and of 10 patients with primary Sjögren's syndrome (primary SS) were examined by light microscopy. Deparaffinized, rehydrated sections of 5 microns were incubated overnight, each with one of 15 different biotinylated lectins. Bound lectins were visualized using avidin-HRP-substrate complexes. Five lectins did not bind to conjunctival cells, and further six lectins bound in an unspecific manner to conjunctival cells of both healthy persons and of primary SS patients. Jacalin lectin bound selectively to goblet cells of all specimens. Peanut agglutinin (PNA) and wheat germ agglutinin (WGA) bound significantly stronger to basal conjunctival epithelial cells of patients with primary SS. This binding pattern may be of diagnostic value.

Experimental mucopolysaccharidosis: preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue.

The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl2 simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.

Attempts of tissue typing from human donor eyes.

A method for HLA-ABC typing using pigmented retinal epithelial and uveal cells of the donor eye is described and the possible implications for corneal grafting are mentioned.

Association of elastin with pseudoexfoliative material: an immunoelectron microscopic study.

Using immunoelectron microscopy, the presence of elastin and tropoelastin was demonstrated in pseudoexfoliative (PSX) material in all its classical sites on the lens capsule, ciliary non-pigment epithelium, iris epithelium and stroma, and conjunctiva. Some variability in binding affinity was seen in different sites, and labelling was more often on the periphery than the center of the PSX fibers. The elastin epitope on PSX material was more sensitive to processing than the remarkably stable epitope on mature elastic fibers. Since neither elastin nor a related component of PSX fibers, elastic microfibrillar protein, is a circulating protein, both are likely to be secreted by local ocular cells. Most of these local cells are not involved in elastogenesis normally, suggesting that an abnormal stimulus or defective regulation of matrix synthesis exists in this disease.

Conjunctival oxygen tension monitoring in emergency department patients.

Conjunctival oxygen tension (PcjO2) was sequentially monitored in 96 medical and surgical patients admitted to the emergency department resuscitation suite during a 6-month period. There were 28 patients with cardiac arrest, 44 with major trauma, and 24 with severe medical problems. A total of 2,392 PcjO2 data points were collected in these patients. In patients with cardiac arrest, PcjO2 showed changes in physiological condition as early as or earlier than measurement of vital signs. Measurement of PcjO2 and the finding of a PcjO2 index (PcjO2/PaO2) less than .5 in normotensive multiple trauma patients allowed rapid detection of hemorrhagic hypovolemia. In critically ill medical patients, low values for PcjO2 were found with hypoxemia as well as in conditions associated with decreased cardiac output and tissue oxygen delivery. These two conditions could be distinguished by measuring PaO2 and calculating the PcjO2 index; a PcjO2 index less than .5 was associated with diminished peripheral perfusion and cardiac output, and a PcjO2 index greater than .5 indicated hypoxemia without any compromise in cardiac output. In the group of critically ill surgical and medical patients included in this study, conjunctival oxygen monitoring provided clinically useful information not available from vital signs and permitted identification of physiological instability associated with abnormalities in peripheral tissue perfusion and oxygenation as early as or earlier than conventional monitoring methods.

Non-invasive conjunctival PCO2 and PO2 monitoring during hyper- and hypoventilation.

In order to evaluate potential applications of non-invasive fiberoptic conjunctival carbon dioxide (Pcj,CO2) and polarographic oxygen (Pcj,O2) sensors, we studied the effects of graded hyper- and hypoventilation on Pcj,CO2 and Pcj,O2 values in dogs. Pcj,CO2 values correlated well with Pa,CO2 (r = 0.95, n = 114); the mean Pcj,CO2--Pa,CO2 gradient was 4 +/- 3 (S.D.) Torr. Both hyper- and hypoventilation resulted in decreased Pcj,O2 values, whereas decreased Pa,O2 was observed only during hypoventilation; thus, the Pcj,O2/Pa,O2 index decreased during hyperventilation but was maintained during hypoventilation. Because both cerebral and conjunctival capillary beds vasoconstrict during hyperventilation, this methodology may assist in the non-invasive monitoring of cerebral oxygenation during cerebral resuscitation and surgery. Non-invasive Pcj,CO2 monitoring, which reflects Pa,CO2 during changes in ventilation, may be used to simplify ventilator management and weaning, as well as guide appropriate timing of arterial blood gas analysis in hemodynamically stable patients.

Noninvasive measurement of tissue carbon dioxide tension using a fiberoptic conjunctival sensor: effects of respiratory and metabolic alkalosis and acidosis.

To evaluate potential clinical applications of a newly developed, noninvasive fiberoptic conjunctival carbon dioxide (PcjCO2) sensor designed to measure continuously tissue PCO2 in a vascular bed supplied by the internal carotid artery, we studied the effects of graded respiratory and metabolic alkalosis and acidosis on PcjCO2 in a hemodynamically stable canine model. Respiratory changes were induced by varying the frequency of ventilation and metabolic changes were induced by incremental infusions of sodium bicarbonate and hydrochloric acid. Continuous measurement of end-tidal carbon dioxide tension (PETCO2) was also performed. During respiratory alkalosis and acidosis, PcjCO2 values correlated well with PaCO2 (r = 0.96, n = 106); linear regression analysis of PcjCO2 vs. PaCO2 produced a slope of 1.01 and a y-intercept of 3.94 over a PaCO2 range of 12 to 76 torr. The mean PcjCO2-PaCO2 gradient was 4 +/- 3 (SD) torr. PETCO2 values also correlated well with PaCO2 (r = 0.91), as well as with PcjCO2 values (r = 0.91). Both PcjCO2 and PETCO2 showed a much weaker correlation with PaCO2 during metabolic alkalosis and acidosis, partly because the variation in PaCO2 was less. Moreover, the PcjCO2-PaCO2 gradient increased during the metabolic portion of the study up to a mean of 10 +/- 8 (SD) torr during metabolic acidosis, implying a build-up and/or lack of washout of CO2 from the conjunctival tissues, despite the normal physiologic range of PaCO2 values. We conclude that in a hemodynamically stable canine model, PcjCO2 and PETCO2 values correlate well with PaCO2 during pure respiratory alkalosis and acidosis; the correlation weakens significantly, however, with metabolic alterations in tissue CO2 levels.

Detection of mucus glycoconjugates in human conjunctiva by using the lectin-colloidal gold technique in TEM. III. A quantitative study in asymptomatic contact lens wearers.

We characterized the mucus glycoconjugates produced by goblet and non-goblet epithelial cells in asymptomatic contact lens (CL) wearers. We employed four lectins (proteins of vegetal origin which specifically recognize glycosidic residues: WGA, PNA, SBA and ConA) conjugated with colloidal gold as ultrastructural marker, at Transmission Electron Microscopy. A computerized quantitative analysis was carried out in order to compare the results from the CL wearers to those from the control patients. Goblet cells produce different amount of glycosidic residues, in particular, a significant decrease in the distribution of sialic acid, N-acetylglucosamine, N-acetylgalactosamine, galactose-N-acetylgalactosamine and mannose was observed. The content of glycosidic residues in the mucus vesicles of the non-goblet epithelial cells appeared unchanged as to the normal situation. We speculate that the CL could possibly contribute to the failure of the tear film stability by altering the production of mucus.

Conjunctival oxygen tension at high altitude.

Transconjunctival oxygen tension (PcjO2) was studied using a hypobaric chamber and during mountaineering excursions. Measurements obtained during acute chamber exposures (15-20 min) at sea level, 1829 m (6,000 ft), 3048 m (10,000 ft), 4267 m (14,000 ft) and return to sea level were (means +/- SEM): 60.1 +/- 2.7, 49.1 +/- 1.8, 38.3 +/- 2.4, 27.4 +/- 1.5, and 61.1 +/- 2.8 mm Hg, respectively (n = 13). The ratio of PcjO2 to arterial blood oxygen tension (PaO2) did not change in a consistent manner between sea level and 4267 m; PcjO2 was 74 +/- 6.9% of PaO2. The 16 subjects participating in the mountaineering phase of the study revealed similar means at sea level and 1829 m (57.4 +/- 2.4 and 46.3 +/- 1.9 mm Hg respectively), but a smaller decrement was observed at 3048 m (43.0 +/- 1.6 mm Hg). The difference between mountain and chamber values may be accounted for by a partial acclimatization to altitude brought about by longer exposure on the mountain excursions. A comparison between PcjO2 and transcutaneous oxygen tension during the chamber study suggests that a greater precision and sensitivity is obtained with measurement of oxygen tension at the conjunctival site. PcjO2 measurement is a non-invasive reflection of PaO2 which is suitable for continuous monitoring during hypoxia studies.

Noninvasive conjunctival oxygen monitoring during carotid endarterectomy.

Conjunctival oxygen tension (PcjO2) was measured continuously during carotid endarterectomy in 15 patients to evaluate its sensitivity in patients receiving shunts. These studies suggest that PcjO2 tracks brain perfusion during periods of carotid artery occlusion. Reduced PcjO2 was clearly demonstrated with systemic hypotension, carotid artery clamping, and carotid shunt obstruction and clamping. Monitoring of PcjO2 is noninvasive, easy to perform, offers no danger to the patient, and allows real-time assessment of the local tissue perfusion. It provides valuable information on the effectiveness of carotid oxygen transport and, in conjunction with arterial blood gas values, expresses carotid artery perfusion relative to systemic oxygen transport. Further investigations using the PcjO2 sensor may define criteria for intraoperative carotid arterial shunting in patients with tenuous cerebral perfusion, and for prompt intervention in patients with deteriorating perfusion prior to the onset of life-threatening cerebral ischemia.

Ocular distribution of keratan sulfates during pre- and postnatal development in rabbits.

Twenty-six monoclonal antibodies (MAbs) developed against rabbit corneal proteokeratan sulfate (PKS), were used to evaluate immunohistochemically the ocular distribution of PKS during prenatal and early postnatal development in rabbits. These MAbs were directed against epitopes located in the keratan sulfate (KS) chains of the proteoglycan (SundarRaj et al., 1985). Staining of cryostat sections of the eyes was carried out using an indirect peroxidase-conjugated technique. Only one of the MAbs reacted with the presumptive corneal region at day 13 or 16 of fetal development. By day 20, more MAbs reacted with the corneal stroma. There were distinct differences, however, in the distribution of the epitopes recognized by the various MAbs. A few of them stained only the posterior region of the cornea, whereas others showed a decreasing staining gradient from the posterior to the anterior region. By day 24, all of the MAbs reacted with the corneal stroma, but some reacted also with the limbal region and with the conjunctival stromal matrix. One MAb also reacted with the conjunctival epithelial layer, but only at this stage of development. Conjunctival staining was more intense at day 28 of fetal development and at day 2 postnatally. KS was not detectable in the conjunctiva of adult rabbits with any of the MABs. These results suggest that although KS synthesis starts at very early stages of fetal development, there are progressive changes in its antigenic structure in specific regions of the cornea and conjunctiva during corneal development.

Clinical, electron microscopic, and monoclonal antibody studies of intraocular epithelial downgrowth.

Epithelial downgrowth developed in three patients following cataract extraction and keratoplasty. Light and electron microscopy of the downgrowth tissue disclosed stratified squamous epithelium, but could not determine whether they were derived from conjunctival or corneal epithelium. The epithelial downgrowth contained cells that were connected laterally by desmosomal junctions and displayed well-defined basement membranes. Surface epithelial cells exhibited myriad microvillous processes scant to moderate mitochondria. In one case, prominent hemidesmosomal junctions were present. Immunohistochemical staining with monoclonal antikeratin antibodies revealed immunoreactivity with AE1, AE3, and AE11 in all cases, but with a cornea-specific antibody, AE5, in only one case. We concluded that in this last case, the epithelial downgrowth appears to have originated from the corneal epithelium.

Increased type-III/type-I collagen ratios in diabetic human conjunctival biopsies.

The ratio of type-III to type-I collagen is measured in human conjunctival biopsies from control and diabetic subjects. The tissue is digested by CNBr and the resulting peptides are quantified by SDS polyacrylamide gel electrophoresis. The peptides used are alpha 1-(I)CB7 and alpha 1-(III)CB8. In control population, of type-III collagen slightly increases with age. In two diabetic populations, (juvenile onset diabetes and maturity onset diabetes), the percentage of type-III collagen is significantly higher than in age-matched control groups. These data plus those previously obtained on genetically diabetic mice indicate that diabetes mellitus affects the expression of interstitial collagen phenotype. Preliminary results on prediabetic subjects suggest the role of genetic factors in such alterations.