Biomedical subjects

H Covington

Publications and source records attributed to H Covington.

4 recordsLinked to original sources

PubMed1986

Development of monoclonal antibodies recognizing collagenase from rabbit PMN; the presence of this enzyme in ulcerating corneas.

Rabbit uterine collagenase was purified from the medium of involuting uterus (1-2 days postpartum) in culture using ammonium sulfate fractionation, DEAE-cellulose, heparin-affinity, and high performance liquid chromatography. The enzyme was purified more than 1600 fold. Hybridoma cell-lines producing monoclonal antibodies were prepared by fusing the spleen cells of mice immunized with the purified enzyme with mouse myeloma cells (Sp2/O-Ag14). The hybridoma cells were selected with HAT medium, cloned, and screened by ELISA. Antibody-producing ascites were prepared by injecting hybridoma cell-lines into the peritoneal cavities of mice. Western-blot analysis indicated that the antibodies recognized a polypeptide having a molecular weight of 52,000. The IgG isolated from the ascites inhibited the enzyme. Indirect immunofluorescent staining demonstrated that polymorphonuclear leukocytes (PMNs) in the superficial layer of alkali-burned corneas contained collagenase, whereas stromal cells and PMNs within the stroma were not stained by the antibodies. Our results suggest that collagenases produced by rabbit PMNs are different from those produced by fibroblasts from cornea. We hypothesize that PMNs in alkali-burned corneas secrete all or most of their collagenases by degranulation at the anterior surface of the cornea, and then continue to migrate into the deeper portion of the stroma.

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PubMed1984

Fibronectin in developing rabbit cornea.

Fibronectin is believed to be important in tissue morphogenesis. We examined the distribution of fibronectin in developing rabbit cornea by immunohistofluorescence. Cryostat sections of cornea from 13, 15, and 20-day-old fetuses, 3-day neonates, and adults were incubated with affinity-purified fluoresceinated guinea pig anti-rabbit fibronectin antiserum (aFN). aFN bound to components within the presumptive stromal region and along the basal surfaces of corneal and lens epithelia during early stages of mesenchymal invasion. At 15 days of gestation, fluorescence was associated with the stromal extracellular matrix of the cornea, the subepithelial zone, and the lens capsule. In the 20-day fetus an intense aFN fluorescence was present along the inner corneal stromal border coincident with the formation of Descemet's membrane. Fluorescence within the corneal stroma appeared as fine lines, restricted to the collagen lamellae, remaining through birth and disappearing in the adult. Although stromal fluorescence disappeared in the adult, Descemet's membrane continued to fluoresce, albeit to a lesser extent. The results of our studies indicate the presence of fibronectin in developing rabbit cornea. Because fibronectin is important to cell adhesion in vitro, and because intercellular and cell-extracellular matrix interactions, including adhesion, are necessary for tissue morphogenesis, our observation suggests that fibronectin plays an important role in corneal morphogenesis.

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PubMed1983

Morphogenesis of rabbit corneal stroma.

Corneas of fetal and young albino rabbits were examined by light and transmission electron microscopy. In addition, DNA and hydroxyproline content were measured in developing stroma. The results were compared with similar data from healing corneas in adult rabbits and from developing corneas of other animal species. In the fetal rabbit, the prospective corneal stroma region contains an unorganized, sparse extracellular matrix until about the 13th day of gestation, when mesenchymal cells and capillaries from the hyaloid vessels move in to form the vascular pupillary membrane, endothelium, and stroma. Stromal growth is due to alteration in the density and morphology of the cell population early in development, along with a sequential thickening and thinning of the whole stroma. These events are similar to those reported in primates, but differ markedly from those reported in avian species. Normal developing cornea and healing adult cornea both involve migration of stomal fibroblasts and deposition of extracellular matrix. Stromal fibroblasts in the rabbit fetus are oriented with their long axis parallel to the corneal surface early in development compared with randomly oriented fibroblasts in the early healing wound of adult rabbit corneas. Although collagen and cell number progressively increase throughout the developmental periods studied, the ratio of cells to collagen is high initially but decreases with development. In contrast, the proportion of cells to collagen in the young scar tissue of adult cornea is low initially, indicating a marked deposition of collagen in comparison to that in the early normal developing stroma. The results suggest that the healing tissue differs from the normal fetal stroma in its coordination of cell population growth with collagen deposition and cellular organization.

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PubMedDate not supplied

Quantitative studies of corneal epithelial wound healing in rabbits.

Corneal epithelial defects are covered rapidly by the movement of adjacent epithelium. However, the mechanism of this tissue movement is poorly understood. In this study, the quantity or cell water, protein, and DNA were determined in healing epithelium to test the hypothesis that cell enlargement contributes to the rapid coverage of the defect. In addition, light and transmission electron microscopy and [3H] thymidine incorporation into epithelial cells were used to determine whether the healing tissue moves as a unit or as individual cells. The quantitative determinations lead us to conclude that healing begins with a dramatic rise in cell water, followed by an increase in cell protein and finally by a gradual increase in DNA. The morphologic and autoradiographic evidence strongly suggests that large corneal epithelial defects in rabbits are covered by the movement of adjacent tissue as a unified, multilayered sheet of cells. Furthermore, the cells appear large than normal with minimal changes in intercellular spaces. We suggest that the increase in cell volume is due to water uptake, which plays an important role in covering the defect by increasing the cells' surface area. Protein is then accumulated, followed by cell proliferation.

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