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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
PubMed1998

Temperature-sensitive interactions between RPE and rod outer segment surface proteins.

Phagocytosis of rod outer segments by the retinal pigment epithelium is distinguished by the two distinct temperature-dependent steps of binding and ingestion. This study was designed to see if retinal pigment epithelial (RPE) plasma membrane proteins interact with ROS plasma membrane proteins at temperatures favoring either binding or ingestion. A modified blot overlay assay was used whereby Western blots of RPE plasma membrane proteins were overlaid with biotinylated ROS plasma membrane proteins. RPE/ROS interactions were detected by streptavidin-HRP and the ECL method at 25 degrees C (ingestion), 15 degrees C (binding), and 4 degrees C (little or no binding or ingestion). Unlabeled ROS proteins served as the negative control. Competition with excess unlabeled ROS proteins were used to test the specificity of the protein interactions. Some protein interactions were somewhat temperature dependent. For example, two RPE plasma membrane proteins (200 kDa and 173 kDa) interacted with ROS plasma membrane proteins at both 25 degrees C and 15 degrees C, but not at 4 degrees C. A strongly labeled protein at 50 kDA protein was present at 25 degrees C but weakly labeled at 15 degrees C and at 4 degrees C. Other protein interaction were more clearly temperature dependent. For example, a 110 kDa RPE protein interacted with ROS proteins only at 25 degrees C. Another RPE protein (55 kDa) interacted only at 15 degrees C. These latter data provide correlations between binding events in the assay and previously described stages of phagocytosis.

Animals↗
PubMed1997

Long-term evaluation of corneal endothelial cell transplantation.

PURPOSE: This report describes the clinical course, refractive changes, confocal microscopic and histological evaluation of corneal endothelial cell transplantation in rabbits with long-term follow-up. METHODS: Transplantation of corneal endothelial cells using a cell/carrier device was performed in 19 rabbits. Clinical evaluation between 1-25 months included slit-lamp examination, keratometry, retinoscopy and surface topography. Two grafts in rabbits with 12 and 24 month survivals were evaluated in vivo by 3D tandem scanning confocal microscopy. The same grafts were then processed for transmission electron microscopy. BrdU labeling of the grafted cells in one transplant was performed in order to distinguish between host and grafted endothelial cells. RESULTS: All grafts cleared and remained clear for an average of one year without signs of rejection or inflammation. Postoperative refraction data and topography of the transplants showed progressive development of myopia and steep corneas compared to the unoperated eyes in each case. Confocal microscopy in vivo demonstrated a regular hexagonal pattern of the transplanted endothelial cells and a thickened Descemet's membrane, which correlated with the light and electron microscopic findings. BrdU labeling of the grafted endothelial cells showed a homogenous labeling of cell nuclei 6 months after the transplantation. CONCLUSIONS: This study demonstrates that corneal endothelial cells grown on a biomaterial can be replaced and remain functional for a long period of time.

Animals↗
PubMed1995

Corneal endothelial wound repair in normal and mitotically inhibited cultures.

BACKGROUND: The aim of the present study was to compare the morphology, proliferative activity and cytoskeletal organization of bovine corneal endothelial cells during wound healing under normal and mitotically inhibited conditions. METHODS: Cell cultures were grown to confluency and incubated with the mitotic inhibitor 5-fluorouracil (5-FU; 2.5 micrograms/ml) followed by a touch wound. Control cultures were maintained without 5-FU. Mitotic activity, F-actin, vinculin, vimentin and connexin 43 localization were evaluated before, during and after wound closure. RESULTS: 5-FU inhibited irreversibly the mitotic activity of corneal endothelial cells during the whole wound healing process. In the presence of 5-FU, a high degree of polymegathism and delay in actin and vinculin redistribution to the cell borders after wound closure was observed. Vimentin and connexin 43 immunolabeling revealed only slight differences between 5-FU-treated and control cultures. CONCLUSIONS: Significant changes in cell geometry and cytoskeletal organization in the amitotic corneal endothelium became manifested only after wounding. These changes may influence cell-cell and cell-matrix interactions as well as functional restoration of the monolayer after wound closure.

Animals↗
PubMed1995

Natural, high-mannose glycoproteins inhibit ROS binding and ingestion by RPE cell cultures.

Previous studies have suggested that a mannose receptor mediates the phagocytic uptake of effete rod outer segments by retinal pigment epithelial cells. In the present study, the effect of adding a soluble ligand for the mannose receptor, horseradish peroxidase, was examined. Cultured retinal pigment epithelial cells from Long Evans rats were preincubated with various concentrations of horseradish peroxidase for 20 min followed by a challenge of FITC-labeled bovine rod outer segments for 3 h. Both counts of total rod outer segments (bound and ingested) and ingested rod outer segments were determined. Rod outer segment uptake was reduced, in a concentration-dependent fashion, by an average of 60% of control values when horseradish peroxidase was added to retinal pigment epithelial cultures. Similarly, total rod outer segment values were reduced to 50% of controls in the presence of at least a 10 micrograms ml-1 horseradish peroxidase concentration. Horseradish peroxidase inhibition of retinal pigment epithelial phagocytic capacity was reversible. Other high mannose glycoproteins, such as invertase, beta-glucoronidase, and ovalbumin, were equally effective in preventing rod outer segment ingestion by retinal pigment epithelial cells. These data further support the hypothesis that a mannose receptor on the retinal pigment epithelial apical surface facilitates phagocytosis of rod outer segments.

Animals↗
PubMed1995

Characterization of rod outer segment plasma membrane proteins which bind to the mannose receptor.

Previous work by our laboratory has demonstrated that rod outer segment (ROS) phagocytosis can be mediated by mannose-receptor dependent activity. This study was designed to probe for potential ligands on the ROS surface which could interact with the mannose receptor during the phagocytic cycle. Solubilized ROS plasma membranes were passed over a mannose receptor-Sepharose column in the presence of CaCl2. Proteins specifically bound to the column were eluted using methyl-D-mannoside and EDTA and characterized by gel electrophoresis, lectin blots, and immunoblots. Silver stained gels of ROS plasma membrane proteins eluted from the mannose-receptor column demonstrated six bands: a major band at 36 kD, identified by monospecific antibodies as rhodopsin, and bands of Mr = 39 kD, 67 kD, 76 kD, 97 kD and 100 kD. Lectin blots of the eluted fractions confirmed that all six proteins in these fractions could bind concanavalin A. In summary, these results showed that rhodopsin and several other mannose-containing glycoproteins on ROS plasma membranes were bound to a mannose receptor column, and thus could serve as ligands for mannose receptor-mediated ROS phagocytosis.

Animals↗
PubMed1994

Transplantation of corneal endothelial cells using a cell carrier device.

Penetrating keratoplasty is currently the only treatment for corneal endothelial dysfunction. Although corneal transplantation has a high success rate, a few problems still remain, such as the limited availability of donor grafts, the change in refraction after penetrating keratoplasty, and the higher chance of immune rejection. In this study, a coated hydrogel lens (Chiron Ophthalmics Inc., Irvine CA, U.S.A.) has been used as a carrier to transplant cultured homologous kitten and rabbit corneal endothelial cells into adult cats and rabbits. The transplantation procedure was the same in both species. Corneal endothelial cells from homologous rabbits or cats were seeded on coated hydrogel lenses and cultured until they reached a complete monolayer with an average cell density of 2,500 cells/mm2. Five weeks before transplantation surgery, corneal endothelial cells were scraped to induce corneal edema. The cell carrier device was then transplanted as follows: a trephine cut (7.7 mm) was made into the stroma, producing an outer corneal plug. The inner cornea was then cut by using a 5.5-mm trephine, and this inner plug was discarded. The implant was inserted and the outer corneal plug was sutured back into place. Corneas cleared completely within 3 days in both rabbits and cats, and stayed clear for an average of 40 days in rabbits and 50 days in cats. The histopathological evaluation of the rejected grafts showed vascularized retrocorneal membrane formation in cats, whereas in rabbits severe cellular infiltration of the stroma with neovascularization occurred without retrocorneal membrane formation.

Animals↗
PubMed1994

Elevation of transforming growth factor alpha in cat aqueous humor after corneal endothelial injury.

PURPOSE: To determine if a scrape injury to cat corneal endothelial cells increases the level of mitogenic proteins such as transforming growth factor alpha (TGF alpha) in aqueous humor. METHODS: Aqueous humor of cats was collected at 0, 2, 6, and 24 hours after wounding the endothelium by contact with a cannula tip. Aqueous humor samples collected from sham-wounded cats served as controls. Aqueous humor samples were analyzed for levels of protein, for mitogenic activity using incorporation of tritiated thymidine by cultures of bovine corneal endothelial cells, and for immunoreactive TGF alpha protein using a specific radioimmunoassay. RESULTS: The average protein level in aqueous humor obtained before wounding was low (0.5 mg/ml), increased 26-fold at 2 hours after injury (13 mg/ml), then progressively decreased at 6 hours (8 mg/ml) and 24 hours (2 mg/ml). Levels of mitogenic activity of aqueous humor samples collected 2, 6, and 24 hours after wounding were 2-fold, 2.5-fold, and 0.6-fold higher, respectively, compared to the level of mitogenic activity measured in aqueous humor collected before wounding (0 hours) or in aqueous humor collected from sham-wounded eyes. TGF alpha concentration in aqueous humor collected before endothelial wounding was low (6.8 ng/ml), increased 14-fold 2 hours after wounding (97.4 ng/ml), then progressively decreased at 6 hours (63.3 ng/ml) and 24 hours (35.5 ng/ml) after wounding. TGF alpha concentrations in aqueous humor collected from sham-wounded eyes at 2 hours (9.5 ng/ml) and 6 hours (5.3 ng/ml) were not significantly different from prewound levels. Detergent extracts of bovine corneal endothelial cells contained substantial levels of TGF alpha immunoreactive protein (20 ng/mg protein). CONCLUSIONS: Wounding of cat endothelium causes a rapid increase in mitogenic proteins in aqueous humor including TGF alpha, which may act by an autocrine mechanism to stimulate endothelial wound healing.

Animals↗
PubMed1993

Isolation of the phagocytic compartment from macrophages using a paramagnetic, particulate ligand.

Isolation of Fc receptor-dependent phagosomes from a macrophage cell line, J774 A.1, was accomplished using antibody-opsonized, paramagnetic beads. Following binding and ingestion of these beads, cells were homogenized in a standard membrane isolation buffer. Phagosomes containing the trapped paramagnetic beads were isolated by subjecting the whole cell homogenate to a magnetic field. The method is extremely simple and the preparation of an enriched phagosome fraction from a whole cell homogenate is rapid and highly selective. The method should provide useful starting material for investigators interested in cytoskeletal involvement in phagocytosis, in kinetic studies of ingestion, in phagosome-lysosome fusion, and in the ability of a particular ligand to initiate phagocytosis.

5'-Nucleotidase↗
PubMed1993

Isolation of plasma membrane domains from bovine corneal endothelial cells.

Corneal endothelial cells are polarized cells responsible for pump, barrier and transport functions, which are carried out by proteins located on the apical or basolateral plasma membrane domain. Apical, basolateral, and total plasma membranes of cultured bovine corneal endothelial cells were isolated to characterize their protein constituents. Apical membranes were isolated by applying charged beads onto the cell monolayer, and harvesting the membranes by washing off the beads. Total cell membranes were isolated in cell/bead suspensions followed by sonication and vortexing. Basolateral plasma membranes were scraped off culture dishes after disrupting cells with distilled water. A variety of marker enzyme assays demonstrated, that the plasma membrane domains could be isolated and separated with relative purity, and with only little contamination by intracellular membrane proteins. SDS-polyacrylamide gel electrophoresis showed approximately 50 protein bands for total plasma membranes, 35 protein bands for apical plasma membranes and approximately 45 bands for basolateral plasma membranes.

Alkaline Phosphatase↗
PubMed1993

Corneal endothelial cell transplantation using Descemet's membrane as a carrier.

Rabbit corneal endothelial cells were transplanted into the right eyes of four New Zealand white rabbits using bovine Descemet's membrane as a cell carrier. Descemet's membranes were dissected from cow eyes, flattened on 36 mm culture dishes, and cut into discs with a 6 mm trephine. Rabbit corneal endothelial cells were seeded onto the discs and maintained in tissue culture conditions for seven days until a complete monolayer with a cell density of 3,000 cells/mm2 was formed. Before transplantation, corneal edema was induced in the host animals by an endothelial scrape wound, which removed the corneal endothelium. Five weeks later, the animals were prepared for transplantation. A corneal button was cut and placed on a dissection table so the host Descemet's membrane could be removed. The donor Descemet's membrane with a complete monolayer of rabbit corneal endothelial cells was placed on the stroma of the corneal button. To keep the donor membrane in place, the button was cauterized at three peripheral points and sutured back into the host eye. Prednisolone sodium phosphate eyedrops and dexamethasone eyedrops and ointment were applied twice daily during the post-operative period. All grafts stayed clear for a period of 12 to 17 weeks. This study shows the potential for using Descemet's membrane as a carrier for corneal endothelial cell transplantation.

Animals↗
PubMed1993

Effects of growth factors on wound healing in serum-deprived kitten corneal endothelial cell cultures.

The influence of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF) I and II on wound healing was investigated in a corneal endothelial system with minimal mitotic activity, using serum-deprived kitten corneal endothelial-cell cultures. After wounding, growth factors were added and wound diameter was evaluated. The DNA synthesis was determined by 3H-thymidine labeling. Wounds did not close in the control cultures grown in serum-free medium without growth factors. The IGF I or II, alone (10 and 100 ng/ml) or added (10 ng/ml) to EGF or bFGF, had no significant effect on wound closure or thymidine uptake. With EGF or bFGF (10 ng/ml), wounds closed after 15 days. Wounds closed after 10 days with EGF or bFGF (100 ng/ml) alone or with the combination of EGF and bFGF (each at 10 ng/ml). Combined EGF and bFGF (each at 100 ng/ml) did not enhance wound closure further. Thymidine uptake was significantly higher in cultures treated with EGF or bFGF (10 ng/ml) than in controls. The uptake could be increased, if both growth factors were combined, but only to the same level achieved with a single factor at 100 ng/ml. This study showed that EGF and bFGF, but not IGF I or II, enhanced wound closure and DNA synthesis in a corneal endothelial cell system that had minimal mitotic activity.

Animals↗
PubMed1993

Enhanced healing of cat corneal endothelial wounds by epidermal growth factor.

PURPOSE: The authors investigated whether healing of cat corneal endothelial wounds could be enhanced in vivo by human epidermal growth factor (EGF). METHODS: EGF was administered in sodium hyaluronate to the anterior chamber of cats after an endothelial touch injury. Control contralateral eyes received sodium hyaluronate alone. At selected times after injury, the corneas were evaluated for thickness, the rate of endothelial wound closure, the endothelial cell density, any variation in cell size, the percentage of hexagonal cells, and endothelial cell mitosis. RESULTS: Two days after injury, endothelial wounds of eyes treated with EGF had healed an average of 65 +/- 4% of the initial 38.5 mm2 wound area; paired control eyes had healed an average of 59 +/- 4% (P < 0.05). Both EGF-treated and control wounds had resurfaced over 90% of the initial wound area on day 4 after injury, and the wounds were completely resurfaced by 7 and 14 days after injury in both treatment groups. On days 4 and 7 after injury, the EGF-treated corneas were 5% and 8% thicker (835 versus 796 microns and 786 versus 728 microns, respectively) than the paired control corneas (P < 0.03). On days 10 and 14 after injury, both EGF-treated and control corneas were 19% and 12% thicker, respectively, than prewound the corneal thickness (621 microns). Seven days after injury, the corneas treated with EGF had an average of 76 +/- 28% more (P < 0.05) endothelial cell nuclei labeled with tritiated thymidine compared with that of the paired control eyes (2472 versus 1543 labeled nuclei). Fourteen days after injury, the central endothelial cell density of EGF-treated corneas was an average of 38 +/- 11% higher than that of the paired control eyes (P < 0.01, 1708 versus 1235 cells/mm2). The percentage of hexagonal cells in the wound area was an average of 14 +/- 4% higher (P < 0.01) than that of the paired control eyes (82% versus 69%), and the coefficient of variation of the cell size for EGF-treated corneas was an average of 31% (P < 0.05) smaller than that of the paired control corneas (0.21 versus 0.29 [standard deviation]/mean cell size). CONCLUSIONS: A single intraocular application of EGF formulated in sodium hyaluronate after an endothelial cell injury significantly enhanced multiple parameters that are closely related to improved endothelial cell regeneration.

Animals↗
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↗