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Biomedical subjects

B J McLaughlin

Publications and source records attributed to B J McLaughlin.

At least 19 recordsLinked to original sources
PubMed1992

Growth of human corneal endothelium on altered Descemet's membrane.

To determine whether Descemet's membrane (DM), which is altered by disease, interferes with endothelial cell growth, healthy human corneal endothelial cells were seeded onto DM from normal corneas and diseased corneal buttons from patients with Fuchs' endothelial dystrophy and pseudophakic bullous keratopathy (PBK). DM was first peeled off the corneal specimens and the endothelial cells removed by trypsinization. A suspension of first-passage corneal endothelial cells (2,000 cells/microliters; obtained from donor eye bank eyes and grown in Dulbecco's minimal essential medium with 10% fetal bovine serum and 1.5% chondroitin sulfate) were seeded on DM. Epidermal growth factor (10 ng/ml) and insulin (1 U/ml) were added to the medium after seeding cells on the DM. The cells attached and flattened within 1 hour and reached confluency in 1 week on normal DM. Cells grown on DM from corneas of patients with Fuchs' endothelial dystrophy also flattened and grew to confluency in 1 week. Cells grown on DM from corneas of patients with PBK did not grow to confluency. Further studies with bovine and rabbit corneal endothelial cells showed similar growth pattern to human cells. These data indicate that DM from corneas of patients with Fuchs' dystrophy does not interfere with the growth of corneal endothelial cells but that DM from corneas of patients with PBK does interfere with cell growth.

Animals↗
PubMed1992

[The pathologically changed Descemet membrane. Cultivation of human corneal endothelium on transformed membrane].

Corneal endothelial diseases are connected with structural and biochemical changes of Descemet's membrane (DM). Little is known about the possible effects of these changed basement membranes. In the present study we investigated the influence of changed DM on endothelium. Human corneal endothelial cells were cultured on DM of healthy corneas as well as corneas with Fuchs' endothelial dystrophy or bullous keratopathy, and cell growth and morphology were compared. On healthy DM, cells formed a confluent monolayer within 4 days. Cells cultured on Fuchs' DM showed a similar pattern. The polygonal cell pattern was interrupted merely in the area of the guttae, which were covered by cell processes alone. Culturing cells proved to be difficult on DM from corneas with bullous keratopathy. Even though the cells attached to the DM, they did not spread but became spindle shaped and rarely formed intercellular contacts. Even after 7 days no confluent monolayer was established. These results indicate that some though not all pathological DM changes interfere with cell growth.

Adult↗
PubMed1991

Comparison of saliva and blood for human immunodeficiency virus prevalence testing.

Testing saliva for the detection of human immunodeficiency virus (HIV) antibodies has many potential advantages for epidemiologic surveillance. A commercial ELISA kit and a standardized in-house immunoblot (IB) system were slightly modified to enhance antibody detection in saliva. Frozen saliva specimens from Toronto Sexual Contact Study participants (including sequential saliva specimens collected during seroconversion) were tested as were fresh saliva samples collected from a population of street-based intravenous drug users (IVDUs). HIV antibody results on saliva were compared with HIV serostatus determined by ELISA and IB on serum or dried blood spots. The overall sensitivity was 98.3% (117/119) for the kit and 99.2% (118/119) for IB; the specificity was 100% (429/429). In the IVDU population, compliance in the voluntary submission of specimens increased from 69% agreeing to provide blood samples to 89% agreeing to provide blood, saliva, or both. Saliva specimens can be easily collected under difficult field conditions with minimal training and provide a valuable alternative to testing blood for HIV-seroprevalence studies.

Cohort Studies↗
PubMed1991

A mannose receptor is involved in retinal phagocytosis.

A 175-kD mannose-specific receptor has been described in macrophages which appears to mediate pinocytosis and phagocytosis. A mannose-specific receptor has also been found on retinal pigment epithelial cells (RPE). Its role was examined in the phagocytosis of photoreceptor outer segments (ROS) by the RPE. All testing was done on cultured RPE cells challenged with fluorescein isothiocyanate (FITC)-labeled ROS for 4 hr at 37 degrees C. Total (internalized and bound) and phagocytized (internalized) ROS were quantified, and the effects of several experimental conditions on ROS phagocytosis were examined. Incubation of RPE cells in the presence of rabbit anti-serum (1:100) raised against the rat alveolar macrophage mannose receptor (anti-Mr) showed a 80% reduction in ROS phagocytosis compared with RPE in the presence of ROS alone. Anti-Mr preabsorbed with purified human mannose receptor protein (4 micrograms) showed no reduction in ROS phagocytosis. Incubation of RPE with preimmune serum showed no reduction in ROS phagocytosis. When FITC-labeled ROS (1.8 X 10(7)) were preincubated with purified mannose receptor, there was a 93% reduction in phagocytosis. Immunoblots of solubilized rat RPE microvillus membranes stained with the anti-Mr showed a single stained band at the 175-kD region, and immunolocalization studies showed specific labeling along RPE microvilli. These results suggest that a mannose-specific receptor is involved in retinal phagocytosis.

Animals↗
PubMed1991

Glycoproteins in the retinal pigment epithelium of normal and dystrophic rats.

The apical membranes of retinal pigmented epithelium (RPE) were isolated from adult, normal (LE), and dystrophic (RCS) rats. The proteins of these RPE subfractions were separated through the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin-binding properties of glycoproteins were examined in western blots through the use of lectin-peroxidase conjugates. No differences were detected between RPE membrane proteins from normal and dystrophic rats in silver-stained gels. However, these two preparations showed significant differences with respect to their binding of the lectins, Lens culinaris (Lentil), Tetragonolobus purpurea (Lotus), and concanavalin A (Con A). In particular, a glycoprotein with a molecular weight of 86 kD in the RPE apical membrane from normal rats bound Lentil, Lotus, and Con A, but in the membrane from dystrophic rats these binding sites were absent or significantly reduced. Another glycoprotein with a molecular weight of 175 kD was recognized by Lotus in the normal membrane preparation but not in the dystrophic RPE membrane preparation. Developmental studies show that these lectin-binding anomalies appear after postnatal day 11 and are, therefore, most likely coincident with eye opening in RCS rats. These results demonstrate that the RPE glycoproteins (86 and 175 kD) are significantly modified in dystrophic rats. The data also confirm previous observations that differences in the oligosaccharide chains, but not the polypeptide chains, of RPE membrane glycoproteins can be detected between normal and dystrophic rats. To the authors' knowledge, this is the first study to correlate developmentally regulated alterations in specific membrane-associated molecules in the RPE of dystrophic rats with the breakdown in phagocytosis that occurs in these rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Isolation and characterization of a mannose receptor from human pigment epithelium.

Recent work demonstrated that a mannose receptor is involved in the phagocytosis of rod outer segments by the rat retinal pigment epithelium (RPE). In this study the binding of soluble mannose-containing ligands by human RPE explants is described. In addition, the authors report the isolation of a mannose receptor from human RPE and describe its relationship to the macrophage mannose receptor. Epithelial explants bound the soluble ligand 125I-mannose bovine serum albumin (BSA) by a mannose-specific process. The protein involved in mannose recognition was extracted from human tissue and purified using ligand-affinity chromatography. The protein that bound to the affinity column had a molecular weight of 175 kD by sodium dodecyl sulfate gel electrophoresis and migrated with the same mobility as the human macrophage mannose receptor. Antibodies directed against the macrophage receptor crossreacted with the mannose receptor from human RPE by immunoblot analysis. Binding specificity studies demonstrated that mannose and mannan inhibited ligand binding to the purified receptor by 65% and 90%, respectively; galactose had no effect. Using immunogold labeling of human RPE cells in explant culture, antimacrophage mannose receptor was localized at the apical plasma membrane. These results suggest that human RPE expresses a mannose receptor on its apical surface (as does the rat RPE) and that this receptor is similar to the human macrophage mannose receptor.

Chromatography, Affinity↗
PubMed1991

Comparison of three HIV antigen detection kits in sequential sera from a cohort of homosexual men.

HIV antigen detection kits are available from a number of commercial sources. Abbott, Coulter, and Du Pont antigen kits were used to test 661 sera collected sequentially from 65 members of the Toronto Sexual Contact Study (TSCS). The sera had been collected at 3-month intervals over 4 years from nine persistently HIV-seronegative men, 14 seroconverters, and 42 seroprevalent participants. Antigen was not detected in any seronegative men. Two of 14 seroconverters were antigen positive in the specimen immediately preceding seroconversion (by all kits). Antigen was detected in 22 of 56 seropositive participants; of these, 16 of 22 demonstrated the emergence of antigen during observation. Discrepancies were noted in the time of detection of antigen (ranging from 3 months to more than 3 years) in nine participants. Although overall concordance among all kits for all specimens appears high (95.4%), when the bias introduced by testing multiple specimens from the same patient is removed, the lower bound of concordance among all three kits is estimated to be 80%. Similarly, after correction, the upper and lower bound of estimates of sensitivity are Abbott 96, 92%; Coulter 88, 63%; and Du Pont 88, 58%. There are significant differences in the performance characteristics of these commercial products for the detection of HIV antigen in serum.

AIDS-Related Complex↗
PubMed1990

The effect of swainsonine on the phagocytosis of rod outer segments by rat RPE.

From studies using inhibitors such as tunicamycin and castanospermine, it has been suggested that plasma membrane glycoproteins may function as receptors in the phagocytosis of rod outer segments (ROS) by the retinal pigment epithelium (RPE). The exact structure of the oligosaccharide side chain of the glycoprotein may not be critical for this process. We have employed another inhibitor, swainsonine, which inhibits mannosidase II, a terminal enzyme in the protein glycosylation pathway, which results in membrane glycoproteins having hybrid-type oligosaccharide chains and fewer complex oligosaccharide chains. We have examined the ability of cultured rat RPE explants to phagocytize fluorescein isothiocyanate (FITC) labelled ROS or latex beads in the presence and absence of swainsonine. A significant (p less than 0.05) reduction in the phagocytosis of FITC-ROS was found between the swainsonine treated (37.7 +/- 4.1%) and untreated (85.4 +/- 2.7%) RPE explants. The nonspecific uptake of latex beads in both swainsonine treated (85.3 +/- 2.4%) and untreated (89.3 +/- 2.0%) RPE explants indicate that the RPE cells retained their ability to phagocytize. Major differences in spectrophotometric analysis of WGA-stained blots were an absence of a peak at 201 kD, a doublet at 86 kD and an overall reduction in all peak absorbances in the swainsonine treatments as compared to the untreated controls. These results suggest that the alterations in RPE glycoprotein formation due to swainsonine alter the ability of RPE to phagocytize ROS.

Alkaloids↗
PubMed1990

Adsorptive and fluid phase endocytosis by cultured rabbit corneal endothelium.

In addition to maintaining corneal transparency via a pump-leak mechanism, the corneal endothelium plays an active role in the transport of certain proteins to supply nutrients for the stroma and to remove metabolites. In order to investigate transcellular transport mechanisms across the endothelium, we exposed cultured rabbit corneal endothelial cells to tracers that are commonly used to describe various forms of endocytosis. The cells were incubated on their apical surfaces in solutions containing different HRP conjugated lectins (concanavalin A and lens culinaris = alpha-D-mannose, alpha-D-glucose; peanut agglutinin = alpha-D-galactose), cationized ferritin (CF), native ferritin (NF), and HRP alone for 5-60 min and processed for EM cytochemistry. At early times, the lectins and CF were seen bound to the apical plasma membrane, thus indicating adsorptive endocytosis. The NF and the lectins in the presence of their competing sugar as well as HRP showed little or no surface binding, thus being markers for fluid phase endocytosis. At later times, large amounts of lectins and CF were located in round, tubular, or U-shaped vesicles of various sizes, large vacuoles, multivesicular bodies, and in other cytoplasmic compartments. Very little or no uptake was observed with NF, or when the lectins were used in presence of their competing sugars. HRP was seen in moderate amounts only in round or oval shaped vesicles. This study suggests that adsorptive endocytic pathways play a major role in transcellular transport through the corneal endothelium, whereas the transport of macro-molecules via fluid phase endocytosis is more limited. In addition, our observations suggest that adsorptive endocytic tracers undergo various intracellular fates and also appear to be transported through the cells at different rates.

Adsorption↗
PubMed1990

Corneal endothelial junctions and the effect of ouabain.

Paired rabbit corneas were perfused in vitro for endothelial permeability (Pac) determination with glutathione bicarbonate Ringer's solution (GBR) and GBR plus ouabain (10(-4) M). Results indicated no difference in Pac between the two groups (3.39 vs 3.67, respectively) despite significantly greater stromal swelling in the group perfused with ouabain. Freeze-fracture microscopy of similarly perfused corneas revealed intact tight junctional complexes in both groups, although the tight junctional complex of perfused corneas appeared less organized than that of freshly enucleated, nonperfused controls. Gap junctions were abundant as observed in freeze-fracture replicas of GBR-perfused endothelium, and appeared to be decreased or absent in ouabain-perfused endothelium. These results indicate that corneal endothelial tight junctions are unaffected by perfusion with ouabain, whereas gap junctions appear to be lost. The permeability and freeze-fracture data reaffirms the importance of tight junctions as permeability barriers and indicates that gap junctions are not of primary importance for maintenance or control of the corneal endothelial barrier.

Animals↗
PubMed1989

A correlative electron microscopic and freeze-fracture examination of cat corneal endothelial wound repair.

In an attempt to create a model for sustained corneal edema in humans, the present study has examined wounded cat corneal endothelium. Small central (7 mm) wounds or large 90 percent debridement wounds were created with an olive tipped cannula and corneas sampled from 1 to 75 days post-wounding were processed for light and transmission electron microscopy and freeze-fracture. In small wounds, wound closure was complete by 14 days and corneal edema was absent. During wound closure, leading edge cell membranes had decreased intramembrane particles, numerous vesicle fusion sites and lacked cell junctions. Endothelium behind the wound margin was multilayered with fragmented cell junctions. After wound closure, endothelium returned to the morphology of non-wounded endothelium except that an abnormal posterior collagenous layer (PCL) was present. Wound closure was greatly retarded in large wounds and corneas remained edematous at 75 days. The morphology of the endothelial cells was similar to that in small wounds except for the presence of large multinucleated cells and a thicker PCL. These large wound findings are similar to those observed in chronically stressed dysfunctional human corneal endothelium and in this animal model may represent a similar response to injury.

Animals↗
PubMed1989

Moderate Fuchs' endothelial dystrophy ATPase pump site density.

The Na+, K+-ATPase pump site density on corneal endothelial cells from Fuchs' endothelial dystrophy corneas has been shown to be drastically decreased in end-stage disease (McCartney et al, Invest Ophthalmol Vis Sci 28:1955, 1987) and significantly increased in the early stages (Geroski et al, Ophthalmology 92:759, 1985) as compared to normal endothelium. In order to provide values for corneas between these two extremes, eye bank corneas from donors with no evidence of corneal edema but with guttata across the extent of the cornea were processed for autoradiography as well as immunohistochemistry. Pump site density was increased compared to end-stage disease but was less than values reported for either functional tissue or early stage disease. Similarly, immunohistochemistry results showed the amount of Na+, K+-ATPase antibody localization to be increased in respect to end-stage disease, but reduced as compared to functional tissue. These results suggest that pump site density on endothelial cells affected with Fuchs' endothelial dystrophy follows a gradual decline towards end-stage disease values as opposed to a sudden sharp deterioration after an initial increase.

Aged↗
PubMed1988

Age-specific prevalence of Epstein-Barr virus antibodies in adult patients with affective disorders.

It has been suggested that prior Epstein-Barr virus (EBV) infection is a necessary condition for the development of affective disorders. To address this issue, the authors performed serologic screening for the presence of EBV antibodies in 60 adult patients seen at an affective disorders investigational unit. Six patients with clinically significant mood disorders, all aged 32 years or younger, had no detectable EBV antibodies. The authors conclude that the onset of clinical mood disorders can occur before primary EBV infection; thus, EBV infection is not a necessary condition for the development of mood disorders.

Adolescent↗
PubMed1988

Mannose 6-phosphate receptors on the plasma membrane on rat retinal pigment epithelial cells.

The retinal pigment epithelium (RPE) phagocytizes the tips of photoreceptor outer segments (OS) during normal eye function. It is not known what ligand on OS is recognized by the RPE for removal from the interphotoreceptor matrix. It is possible that a sugar residue on a cell surface glycoconjugate of either the OS or RPE mediates the phagocytic interaction. Pinocytic experiments with a soluble mannose 6-phosphate ligand (125I-labelled mannosidase) showed that similar quantities of ligand were bound by RPE explants from Long Evans rat retinas and from Royal College of Surgeons (RCS/p+) rat retinas known to be defective in the phagocytosis of OS. The addition of mannose 6-phosphate reduced the total counts of bound alpha-mannosidase by 23% in both normal and dystrophic RPE explants. Mannose 6-phosphate receptors were visualized on normal and dystrophic RPE plasma membranes by immunocytochemical techniques. Further, phagocytosis was studied using phosphomannan-coated beads as phagocytic particles. Dystrophic RPE phagocytized phosphomannan-coated beads by a mannose 6-phosphate specific mechanism as shown by a significant reduction in the number of these coated beads taken up in the presence of the competing sugar. In contrast, normal RPE showed no uptake of phosphomannan-coated beads. Our findings indicate that a mannose 6-phosphate receptor is on the apical plasma membrane of rat RPE. This receptor may not be involved in normal OS phagocytic recognition, but may function in the trafficking of lysosomal enzymes by RPE cells.

Animals↗
PubMed1988

Expression of mannose receptors for pinocytosis and phagocytosis on rat retinal pigment epithelium.

We report here the presence of a mannose-specific receptor on apical membranes of rat retinal pigment epithelial (RPE) cells. For pinocytic studies, 125I-Mannose-BSA (125I-Man-BSA) was incubated with RPE explants from normal (Long Evans) and dystrophic (pigmented RCS) rat retinas. Normal RPE bound 36.1 ng of ligand and, in the presence of mannan competitor, the amount bound was 18.3 ng. In a similar assay, total ligand uptake by dystrophic RPE was 25.9 ng with 9.8 ng specific for mannose recognition. Comparing the amounts of ligand bound, dystrophic RPE recognized 55% of the amount recognized by normal RPE. The presence of the mannose receptor was localized on both normal and dystrophic RPE apical plasma membranes by autoradiographic techniques using 125I-Man-BSA. Normal RPE showed a greater number of silver grains present at the apical cell membrane as compared to dystrophic RPE. Silver grains were significantly reduced when incubation with the ligand was carried out in the presence of a mannan competitor. Further, in phagocytic studies, latex beads coated with mannan were used as phagocytic particles. Normal RPE phagocytized 4.52 mannan-beads per cell profile by a mannose-specific mechanism, whereas dystrophic RPE did not recognize mannan-beads. Our data suggest that RPE cells express surface receptors which recognize both soluble and particulate mannose ligands. The pinocytic and autoradiographic studies suggest that normal RPE binds more soluble ligand than does dystrophic RPE. If the mannose receptors mediate both pinocytosis and phagocytosis, a possible reduction in number of soluble mannose binding sites on the dystrophic RPE may be related to the diminished phagocytic recognition of particulate ligand by the dystrophic RPE.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Quantitative freeze-fracture and filipin-binding study of retinal pigment epithelial-cell basal membranes in diabetic rats.

Breakdown of the blood-retinal barrier in diabetes may be related to alterations in the retinal pigment epithelial (RPE) cell layer. Morphological studies suggest increased permeability of diabetic RPE plasma membranes, and proliferation and flattening of the RPE basal infoldings have been observed in diabetic animals. In order to determine whether these phenomena are associated with changes in membrane protein or sterol composition, we have used quantitative electron-microscope freeze-fracture and filipin-binding techniques to study the RPE basal membrane in streptozotocin diabetic and 3-O-methyl glucose control rats. Perfusion-fixed retinas were processed for freeze-fracture and filipin-binding analysis. Filipin, a polyene antibiotic, binds specifically to 3-beta-hydroxy-sterols to produce membrane deformations recognizable by freeze-fracture. These analyses revealed an 11% increase in the density of intramembrane particles within the cytoplasmic (P-face) leaflet in diabetic rats as compared with the controls (P less than 0.01, t test). The increase occurred primarily in 6-9-nm particles, while smaller particles were decreased (P less than 0.001, chi-square test). Filipin binding was the same in both groups. These results suggest that alterations in intrinsic membrane proteins may contribute to permeability and surface area changes in the diabetic RPE but that RPE membrane sterols are not affected by diabetes.

Animals↗