Characterization of human lens antigens by crossed immunoelectrophoresis

Crossed immunoelectrophoresis and fused rocket electrophoresis were used to examine fractions of human lens homogenates obtained by ion exchange (DEAE) and gel (G-200) chromatography. The methods were found useful for identifying the various antigens found in each fraction, to evaluate the purity of the fractions, and to compare both qualitatively and semi-quantitatively, the antigenic content in the various fractions. It is suggested that these immunological techniques may provide the basis for an extended nomenclature of lens crystallins.Considerable differences were found between fractions from lenses of young individuals and from those of old individuals. The fractions from young lenses were fairly homogeneous while those from old lenses were heterogeneous. The DEAE fraction 0·4 m from old human lenses contained, in addition to α-crystallin, considerable amounts of β- and γ-crystallins. It is suggested that this, at least in part, explains the presumed increase in α-crystallin content of human lenses with age.The α-crystallin preparations obtained by ion exchange and gel chromatography were equally pure. The purest β-crystallin preparations were obtained by ion exchange chromatography while the purest γ-crystallin preparation was obtained by gel chromatography.     

Inhibition of collagenase activity by extracts of bovine ocular tissues

Bovine eyes were dissected and separate pools of lens, lens capsule, cornea and vitreous were extracted in guanidine, subjected to ultrafiltration, and examined for their effects on collagenolytic activity. Although lens extract was not inhibitory, the cornea and vitreous both contained inhibitors of collagenase. More inhibition was present in the filtrate of the vitreous than in the retentate, whereas the total amount of inhibition in the cornea was distributed almost equally between the two fractions. The inhibition observed was dose dependent. The partially purified inhibitors from cornea and vitreous blocked the activity of human skin and tadpole back skin collagenases, but they failed to inhibit the bacterial (Clostridium histolyticum) collagenase. The inhibitor was stable to heating to 60 degrees for 30 minutes and to trypsinization.     

Human ocular mucus. Origins and preliminary characterisation.

A technique for collection of human ocular mucus was developed, and the solubility of the crude mucus clot in a variety of solvents, reducing agents and detergent solutions was assessed. Reduced and unreduced tears and mucus were separated by polyacrylamide gel electrophoresis on slab gels, and their mucous glycoprotein and other protein components identified by Coomassie Blue and periodic acid-Schiff staining. The principal mucin complex GP1 (mol. wt.>2×10⁶) and its subunit GP3M (mol. wt. about 200 000) were detected in unreduced mucus, while reduction of disulphide bonds gave rise to GP2 (mol. wt. about 1·3 × 10⁶). Human GP2 and GP3M each contained a high proportion of carbohydrate, but there was considerable variation among individual donors. Rerunning of the GP2 band isolated from gel gave GP2 and also a GP3M band. Immunofluorescent studies using rabbit antibody raised against human GP2 showed the site of origin of GP2 to be exclusively the conjunctival goblet cells and not lacrimal tissue. A single mucous glycoprotein GP3T, also about 200 000 mol. wt., was found in tears, but did not cross-react with anti-GP2 antibody.     

O-glycosidic linkage in glycoprotein isolates from human ocular mucus

Alkaline beta-elimination and sodium borohydride reduction were used to study the O-glycosidic linkage of N-acetylgalactosamine to seryl and threonyl residues in the high molecular weight crude glycoprotein fractions isolated from human ocular mucus. Pure mucins, BSM and OSM, were used as models. Data are presented for the existence of such O-glycosidic linkages. It was estimated from sodium borohydride reaction that at least 22% of the total peptide bonded hydroxyamino acid residues are linked O-glycosidically. These findings, as a continuation of our previous work on the isolation and chemical characterization of human ocular mucus, provided additional evidence of the mucin nature of the glycoprotein isolates.     

Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatricial pemphigoid using lectin probes and histochemical techniques

Conjunctival biopsies from patients with cicatricial pemphigoid affecting the conjunctiva and patients undergoing cataract surgery (normal conjunctiva) were snap-frozen, cryostat sectioned and incubated with fluorescein-conjugated lectins; peanut agglutinin (PNA), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and succinylated wheat germ agglutinin (S-WGA). Controls consisted of preincubating the lectins with the appropriate blocking sugars before applying the lectins to the sections. PNA and HPA stained the mucus granules contained in the conjunctival goblet cells but did not stain mucus or glycocalyx at the ocular surface distal to the goblet cells. Native WGA and S-WGA had high affinities for conjunctival goblet cells and the apical epithelial cell layers. Native WGA stained mucus and glycocalyx at the ocular surface. This staining of the ocular surface by WGA was confirmed at the transmission electron microscopic level using WGA conjugated to ferritin. Cicatricial pemphigoid patients in this study had reduced numbers of goblet cells; however, those goblet cells which were observed in cicatricial pemphigoid conjunctiva stained positively with HPA, PNA, WGA, and SWGA as did goblet cells in normal conjunctiva.     

Scanning electron microscopic study of Dacron degradation by ocular tissue extracts.

A scanning electron microscopic study showed that the surface of Dacron thread became significantly roughened after treatment with bovine iris-ciliary body extracts. The Dacron-degrading factor in the extract was nondialysable, heat-labile, and active at an acidic pH, suggesting that lysosomal enzymes may be a factor in this phenomenon. Bovine extracts from the iris, ciliary body, and sensory retina degraded the surface of Dacron most substantially, while the Dacron surface was moderately digested by extracts from the cornea and retinal pigment epithelium. Lenticular and choroidal extracts did not affect the Dacron surface. Possibly the factor that degrades Dacron may be different from that which affects nylon, and the Dacron suture may not be preferable for corneal surgery.     

Isolation and preliminary characterization of tear prealbumin from human ocular mucus

Tear prealbumin was purified from crude tear prealbumin previously isolated from the saline soluble human ocular mucus. Purification was achieved by further column chromatographies on DEAE Sephadex A-25 and Sephadex G-75. Preliminary characterization included amino acid analysis, gel electrophoresis, and isoelectric focusing. Unlike serum prealbumin, the purified tear prealbumin showed a predominance of acidic residues and a trace amount of tryptophan. It exhibited polymorphic nature, with pI values of 4.8 and 4.9. The possibility of a tear prealbumin/retinol complex was also examined. The protein was found to incorporate with 3H retinol. The 3H retinol-incorporated tear prealbumin did not exhibit the characteristic UV spectrum of retinol; however, it did display emission and excitation fluorescence spectra at high concentrations similar to serum retinol-binding protein.     

Association of serum albumin with tear prealbumin in human ocular mucus

Tear prealbumin previously isolated from the saline-extractable human ocular mucus was further examined for microheterogeneities using crossed-immunoelectrophoresis through development with antiserum to the isolate. The isolate was examined following the final two stages of its chromatographic purification by DEAE ion exchange and Sephadex G-75. The crossed-immunoelectrophoretic pattern of the tear prealbumin isolate, recovered following DEAE ion exchange chromatography, indicated the existence of two types of minor degrees of binding between tear prealbumin and serum albumin. One type appeared to be tightly associated while the other was interpreted as weakly bound, where serum albumin was separable from tear prealbumin in an electric field. The total association of serum albumin with tear prealbumin in this isolate was estimated to be about 3-7%. Crossed immunoelectrophoresis of the isolate after its final stage of purification on Sephadex G-75 chromatography showed the absence of the weakly bound serum albumin. However, the tight tear prealbumin/serum albumin association remained, estimated to be 1-3%.     

Fractionation and partial characterization of macromolecular components from human ocular mucus

Crude human ocular mucus was extracted with 0·154 m-NaCl to separate soluble protein components from mucus. Small amounts of lipoglycoprotein of high molecular weight, as well as twelve plasma proteins, were detected in the soluble extract by gel filtration and immunodiffusion studies. After the NaCl extraction, the remaining mucus residue was further extracted with 6 m-urea-0·2 m-Tris-phosphoric acid buffer. From this portion of soluble extract, a relatively larger amount of lipoglycoprotein of high molecular weight, as well as a lower molecular weight fraction containing eight detectable plasma proteins, were both isolated by gel filtration. The glycoprotein moieties of the lipoglycoproteins of high molecular weight had similar chemical composition. Both contained approximately 40–43% protein and 57–60% carbohydrate, giving a carbohydrate-protein ratio of 1·30 to 1·48. Fucose, galactose, N-acetylhexosamine and N-acetylneuraminic acid comprised about 423–516 residues per 1000 amino acid residues, while serine and threonine constituted about 285–299. All analyses indicated mucin-like character in the lipoglycoproteins of high molecular weight.Plasma proteins constituted approximately three-fifths of the macromolecular components in ocular mucus. These proteins also appeared to be in complexes with lipids, but to a much lesser extent than the high molecular weight fractions. The relevance of present findings to the structure and composition of precorneal tear film is discussed.     

Impaired neural conduction in crossed visual pathways in patients with ocular hypertension

PURPOSE: To evaluate the neural conduction along crossed and uncrossed visual pathways in patients with ocular hypertension (OHT). METHODS: Fifteen patients (mean age 59.1+/-6.8 years) with OHT (IOP>22 mmHg, Humphrey 24-2 with mean deviation [MD]>-2 dB) were enrolled. They were compared to 15 age-matched controls. In OHT patients and control subjects, visual evoked potentials (VEPs) were recorded using full-field checkerboard patterns (the check subtended 15' of visual arc; contrast 80%) reversed at 2 Hz. VEP responses were simultaneously recorded in the homolateral visual cortex (HC) and in the contralateral visual cortex (CC), with respect to the stimulated eye. RESULTS: In OHT patients, VEP P100 implicit times observed in HC and CC were both significantly delayed (analysis of variance, p<0.01) when compared to those of controls, and, in particular, longer in CC than in HC. The interhemispheric differences (ID: P100 implicit time in HC - P100 implicit time in CC) were significantly higher in OHT patients than controls (-3.16+/-1.80 msec and 1.16+/-1.04 msec, respectively, p=0.001). In OHT patients we observed an MD hemifield difference (difference between nasal and temporal MD values) higher than in controls (-0.82+/-0.80 dB and 0.04+/-1.03 dB, respectively, p<0.01) and significantly correlated with the ID (r: 0.836, p<0.001). CONCLUSIONS: The observed asymmetry in the bioelectrical cortical responses and in the visual hemifield parameters suggests that crossed visual pathways could be impaired earlier than uncrossed visual pathways in OHT patients.     

Immunological study of proteins and mucosubstance in saline soluble human ocular mucus

Proteins and mucosubstance of the saline extract of human ocular mucus were studied by immunological analysis. A minor study was made with human tears for comparison. Immunoelectrophoresis of proteins from these two sources consistently revealed similar characteristic gel patterns. Proteins were found as the major constituents of both samples. However, more mucosubstance was present in the saline extract of human ocular mucus than in tears. Seventeen proteins were identified in the mucus extract. Albumin, IgA, and lactoferrin appeared to be the three major proteins, while lysozyme, lactoferrin, tear prealbumin, and ocular mucoisolate were tear and ocular mucus specific. Although saline soluble mucoisolate is complex in structure, it seemed to resist electrical dissociation, producing only one major precipitation line along with a line of IgA during immunoelectrophoresis. The ocular mucoisolate accounted for about 12% of the saline extractable proteins of human ocular mucus.     

Studies of ocular retinoblastomas with immunoperoxidase techniques

15 cases of ocular retinoblastomas, 10 differentiated and 5 undifferentiated, were studied with the following antibodies: neuron-specific enolase, S100, and tubulin. All cases turned out negative with S100 and tubulin labeling. The well-differentiated retinoblastomas were NSE-positive, and only 2 of the 5 undifferentiated retinoblastomas were positive for the same antibody. The observed results let us conclude that S100 and tubulin are not useful in diagnosing this entity and that enolase, although it showed its value in the differentiated retinoblastomas, did not have the same effect in the undifferentiated retinoblastomas, as it was negative in 3 out of 5 cases.     

A review of ocular blood flow measurement techniques

Several major eye diseases may be characterized by deficits in ocular blood flow. Maintaining adequate nutrient delivery to the choroid, retina, and optic nerve head may prevent cellular damage and loss, complementing efforts to provide neuroprotection. The ability to quantify deficits in ocular blood flow has increased in recent years with the introduction of several new techniques. 1) Scanning laser ophthalmoscopic fluorescein angiography allows the measurement of bulk retinal flow and macular capillary transit rates. 2) Scanning laser ophthalmoscopic indocyanine green angiography measures choroidal perfusion in selected areas near the optic nerve head and macula. 3) Confocal scanning laser Doppler flowmetry permits quantitation of retinal capillary perfusion. 4)Color Doppler ultrasound imaging measures flow velocities in the ophthalmic, central retinal, and short posterior ciliary arteries. While these techniques represent a major advance, significant issues regarding blood flow and ocular disease remain. First, the hemodynamic characteristics of the disease state need further definition. Second, the blood flow effects of current and future treatments must be established. Third, improvements in ocular blood flow techniques are required, both to increase anatomic precision and to allow non-invasive measurements during times of potential ischemic risk such as sleep. These developments could enhance prevention, diagnosis, and treatment of several major eye diseases.     

Local anaesthetic techniques and pulsatile ocular blood flow

AIM: To compare pulsatile ocular blood flow (POBF) and intraocular pressure (IOP) between eyes of patients receiving either peribulbar (with and without balloon compression) or subconjunctival local anaesthesia (LA). METHODS: 30 eyes of 30 patients undergoing cataract surgery by phacoemulsification were investigated in a study of parallel group design. Ten patients had peribulbar LA and 10 minutes compression with a Honan's balloon (group A). A further 10 patients who received peribulbar LA alone (group B) acted as controls for the effects of balloon compression. Ten other patients were given subconjunctival LA (group C). POBF and IOP were measured using a modified Langham pneumatonometer. Three measurements were made in each eye, the first recording immediately before LA, the second 1 minute after, and the third 10 minutes after LA. RESULTS: No significant change in POBF or IOP was recorded in eyes receiving subconjunctival LA. In the peribulbar groups (A and B), there was a drop in median POBF of 252 and 138 microl/min respectively 1 minute after LA, which was statistically significant in both groups (p<0. 01). By 10 minutes, POBF tended to return to baseline levels, but remained significantly reduced in group B (p<0.05). In addition, there was a significant (p<0.05) reduction in IOP (mean drop of 4.82 mm Hg) in group A following peribulbar LA with balloon compression. CONCLUSIONS: POBF was significantly reduced after peribulbar LA but was unchanged after subconjunctival LA. Balloon compression reduced IOP and improved POBF following peribulbar LA. The findings may have clinical implications in patients with compromised ocular circulation or significant glaucomatous optic neuropathy.     

Characterization of human ocular mucin secretion mediated by 15(S)-HETE

PURPOSE: The eicosanoid 15-(S)-hydroxy-5,8,11,13-eicosatetraenoic acid [15(S)-HETE] is reported to stimulate mucin production in both airway and ocular surface epithelia. The current study was undertaken to evaluate the effects of 15(S)-HETE on secretion of specific ocular mucins by human conjunctiva. METHODS: Segments of human bulbar conjunctival tissue were incubated with 15(S)-HETE (1-1000 nM) for 30 minutes at 37 degrees C. Secretion of human ocular mucins MUC1, MUC2, MUC4, and MUC5AC into the incubation media was measured by dot-blot immunoassay using antibodies directed to unique mucin polypeptide epitopes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were used to verify the specificity of anti-mucin antibody binding and to investigate the presence of MUC1 mucin in human tears. RESULTS: 15(S)-HETE (10(-8)-10(-6) M) stimulated secretion of conjunctival mucins in a concentration-dependent manner. Significant increases in total mucin secretion were observed at 10(-7) M 15(S)-HETE with a maximum response (>50% increase above controls) at 10(-6) M. Results of immunoassays showed that 15(S)-HETE differentially stimulates secretion of MUC1 mucin with no detectable effects on MUC2, MUC4, or MUC5AC release. Western analysis of tear samples from human volunteers indicated that MUC1 is a component of the preocular tear film. CONCLUSIONS: The results demonstrate that 15(S)-HETE is a selective secretogogue for MUC1 in isolated human conjunctival tissue. Although the biochemical mechanism(s) and cellular origins of MUC1 secretion remain to be established, the ubiquitous expression of MUC1 in corneal and conjunctival epithelia and its presence in human tears suggest that secreted MUC1 may contribute to the mucin layer that coats and protects the ocular surface.     

Characterization of acute and delayed ocular lesions induced by sulfur mustard in rabbits

PURPOSE: To establish an experimental model for sulfur mustard-induced acute and delayed ocular lesions in rabbits. METHODS: Rabbit eyes were exposed to sulfur mustard (HD) vapor (370, 420 microg/l) for a period of two minutes. A three months follow-up study was carried out, based on the evaluation of clinical, biochemical and histological parameters. RESULTS: HD exposure initiated typical clinical symptoms within 2-6 hrs, characterized by eye closure, eyelid swelling, conjunctival hyperemia, corneal erosions and inflammation. The clinical signs were significantly dose-dependent and reached a peak at 24--72 hrs post exposure. Biochemical evaluation of the aqueous humor exhibited an inflammatory reaction and oxidative stress at 4 hrs after exposure, subsiding at 28 hrs after exposure. Histological examination of corneas at 48 hrs revealed epithelial denudation and marked stromal edema, accompanied by cellular infiltration. Epithelial regeneration started after 72 hrs, and recovery was almost completed within 1--2 weeks, depending on the HD dose. A second phase of pathological processes started as early as two weeks post exposure and was characterized by corneal edema, opacity, recurrent erosions and neovascularization. The delayed injuries were found in 25 and 40% of the eyes respectively, and when appearing, were more severe than the initial ones. CONCLUSIONS: The development of HD-induced ocular lesions in rabbits is similar to the lesions described in human casualties. Quantitative analysis of the various clinical parameters emphasizes the contribution of each tissue to the overall toxic picture. Our experimental model is useful for studying the pathological mechanisms of HD-ocular lesions, and may serve for testing potential therapies.