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Extracellular matrix (ECM) is an important mediator of endothelial functions such as adhesion, spreading, migration, proliferation, and maintenance of differentiated functions. Attachment of cultured cells to tissue culture polystyrene (TCPS) is dependent on vitronectin which adsorbs onto the surface from the serum in the culture medium. Vitronectin (VN) will adsorb efficiently to TCPS even if the latter has been coated with another matrix molecule and blocked with albumin. This means that studies of the interactions of cells with individual coated ECM molecules will be confounded by the presence of adsorbed VN if serum is present in the culture medium. In this study, the adhesion, spreading, growth, and output of endogenous matrix molecules by bovine corneal endothelial (BCE) cells were measured on five different matrix substrates using medium which had been depleted of vitronectin to avoid such confounding effects. The same cell adhesion and spreading maxima were achieved on vitronectin, fibronectin (FN), laminin (LM), and types I and IV collagen (col I, col IV). The coating concentrations required to achieve these maxima, however, differed among the substrates, LM needing considerably higher concentrations than the other substrates for both maximal adhesion and spreading and FN needing higher concentrations for cell spreading. When cells were continuously passaged on each of the five substrates coated at concentrations optimal for cell spreading, no differences in cell proliferation rates or cell morphology were observed. Significant differences, however, were observed in the subcellular output of endogenous matrix molecules (FN, LM, col IV, and thrombospondin) between the different substrates. Col I was a poor substrate for the production of all ECM molecules tested over the 10 passages of the experiment, whereas col IV was a consistently good substrate. LM and FN substrates displayed differential effects on the output of different ECM molecules. VN was unique in that BCE cells at early passage on this substrate produced high levels of endogenous matrix molecules, whereas with continued passage on this substrate, a progressive decline in ECM secretion was observed. These results show that incorporation of individual molecules into the ECM by BCE cells in culture is significantly affected by the nature of the substratum. They further suggest that passage of endothelial cells in media containing serum (which results in coating of VN onto the substrate) may result in a progressive reduction of ECM output. Copyright 1993, 1999 Academic Press

Reciprocal hemagglutination inhibition titrations were carried out with viruses and antisera of eight field strains of the A3 subtype of influenza A, covering the period from 1968 to 1975. The earlier strains (1968 through 1972) showed asymmetric cross-reactions, with antisera exhibiting more cross-reactions with antecedent strains than with subsequent ones. The later strains, although all were asymmetrically cross-reactive with earlier strains, tended to exhibit distant and variable cross-reactions with each other. The numbers and average affinities of antibody molecules capable of taking part in cross-reactions were calculated from equilibrium filtration experiments. It was found that all the antibody molecules in sera raised against the late strains could combine with earlier viruses, but with reduced affinity. Conversely, only a subset of the antibody molecules in sera raised against early strains could combine with later viruses. The results are discussed in the light of different theories concerning the nature and number of antigenic determinants on the hemagglutinin molecule. They support the existence of a single antigenic area to which all antibody molecules are directed, with differing affinities, rather than the existence of both “common” and “specific” determinants. Thermodynamic measurements on the homologous antigen-antibody reactions indicated that combination was mostly entropy driven. This suggested hydrophobic interaction as the mechanism of combination, i.e., that the complementary regions of antigen and antibody were made up largely or entirely of amino acids with hydrophobic side chains. There was no statistical difference in the magnitude of the entropy term (i.e., the average firmness of binding) among the different virus strains.

This paper describes the biochemical characteristics and immunostaining properties of four monoclonal antibodies (MAbs) raised to the extracellular matrix of bovine corneal endothelial cells in culture. All four antibodies labelled molecules in frozen sections of bovine cornea. MAb A15 labelled the collagen lamellae of the stroma. Immuno-electron microscopy located the label between rather than over the collagen fibrils. Antibody label was removed by collagenasa treatment of the stroma. This antibody bound to several denatured collagen types in ELISAs and Western blots, the common epitope being located close to the collagenasa binding site. A15 immunoprecipitated native polypeptides of 173 kDa and 150 kDa from the conditioned medium of corneal endothelial cells. Amino acid analysis of the 150 kDa molecule showed broad similarity to bovine type VI collagen although there was no immunological cross reactivity. The binding of this antibody in the corneal stroma may be to a collagen type VI-like molecule. MAb A70 bound to a collagenase sensitive molecule in corneal endothelial cell extracellular matrix in vitro, and to basement membranes in vivo. It showed staining typical of type IV collagen in frozen sections of cornea. MAbs A67 and A49 both labelled flexuous fibre-like structures in the cornea, which appeared to wrap around the fibrillar collagen lamellae. The molecule labelled by A67, of 86 kDa was sensitive to collagenase treatment of endothelial cell conditioned medium, yet totally resistant to collagenase treatment of the cornea, which completely removed the fibrillar collagen from the stroma. In the endothelial extracellular matrix, this antigen was resistant to all proteases tested and required severe denaturing conditions to remove antigenic activity. Amino acid analysis did not yield the high proportion of glycine typical of collagens, so if collagenous sequences occur they must be relatively short. The molecule labelled by A49 was a 51-kDa protein, of similar amino acid composition to antigen 67, and showed a similar distribution in frozen sections of bovine cornea. There was, however, no immunological cross reactivity between the two. Unlike antigen 67, antigen 49 was not sensitive to collagenase under any conditions arid was very sensitive to protease treatment of file endothelial extracellular matrix. Immuno-electron microscopy showed labelling with both these antibodies between the collagen fibrils in the stroma. We postulate that these two molecules may be involved in stabilising the lamella structure of the corneal stroma. Copyright 1994, 1999 Academic Press

Human bone-derived cells are known to attach and spread on surfaces which have been precoated with fibronectin, but the contributions made by specific domains of the molecule have not yet been defined. Here we refer to the osteoblast-like cells as human bone cells. We have determined the relevance of separate regions of fibronectin, particularly the heparin-binding region, for the initial attachment and spreading of these cells. Human bone cells attached to fragments from each of the cell- and heparin-binding regions of fibronectin, but failed to attach to a fragment from the gelatin-binding region. Bovine corneal epithelial cells, which were included as an example of an alternative primary cell strain, attached to the cell-binding fragment but showed no specific shortterm attachment to the heparin or gelatin-binding fragments. Monoclonal antibody MAb17, which binds to the cell binding region of fibronectin, partially inhibited the attachment of both human bone cells and corneal epithelial cells to intact fibronectin when present at 50 µg/ml and reduced human bone cell attachment to the cell-binding region fragment of fibronectin. Monoclonal antibody, MAb 32, which binds to the heparin-binding region of fibronectin, failed to inhibit attachment of the human bone cells to fibronectin but reduced the attachment of these cells to the heparin-binding region fragment. Heparin and chondroitin sulphate were able to inhibit human bone cell attachment to the heparin-binding fragment of fibronectin but had no effect on their attachment to intact fibronectin or the cell-binding region of fibronectin. Immunofluorescent staining and confocal microscopy showed extensive spreading and actin filament formation when human bone cells were cultured on intact fibronectin. Cells cultured on the heparin-binding fragment showed only minimal spreading coinciding with less extensive actin filament organisation. On the cell-binding fragment of fibronectin more spreading was seen than on the heparin-binding fragment but it was not as extensive as on intact fibronectin. Taken together, these data suggest that human bone cells, unlike bovine corneal epithelial cells, have an attachment mechanism for the heparin-binding region of fibronectin. Attachment to this region is probably mediated by cell surface proteoglycans. However, interaction with the cell-binding domain is required for effective cell spreading of human bone cells on fibronectin during the first 90 minutes after seeding into culture.

The initial attachment and spreading of endothelial cells from human umbilical artery onto type i collagen, type IV collagen or gelatin substrata was shown to be enhanced by inclusion of serum in the culture medium. To test whether this serum effect was mediated by adsorption of serum fibronectin or vitronectin onto the collagen, these adhesive glycoproteins were selectively removed from the serum prior to addition to the culture medium. The stimulatory effect of serum on human endothelial cell spreading on collagens I and IV was also observed with serum from which either fibronectin or vitronectin, or both, had been selectively removed. The stimulatory effect for cell spreading on gelatin was diminished by selective removal of serum fibronectin, but unaffected by removal of vitronectin. Hu man endothelial cell attachment and spreading onto tissue culture plastic was abolished by removal of vitronectin from the serum in the culture medium. These results emphasize that the native structure of collagens is required for serum-enhancement of human endothelial cell attachment and spreading on native collagen types I and TV, and show that on these substrata the stimulated adhesion and spreading are not dependent upon adsorption of serum fibronectin or vitronectin onto the collagen substratum.

A panel of monoclonal antibodies (mAbs) to bovine fibronectin (FN) is described which modulates either heparin binding or cell adhesion to FN, or both. A combination of competitive exclusion and binding to proteolytic fragments identified epitopes in the Hep II, Hep III/l and CBF (cell binding fragment) regions of FN. mAb A17, which bound to the CBF region, strongly inhibited the cell adhesion of BHK-21 fibroblasts, primary corneal fibroblasts and endothelial cells, and NM4 mammary adenocarcinoma cells, to FN at mAh concentrations as low as 1 μg/ml. This mAh was not so effective at inhibiting the adhesion of Bib mouse melanoma cells. Adhesion of B16 cells to FN was more sensitive to inhibition by mAbs binding to Hep H (A2, A9, A32, A35). Of these, A32 and A35 significantly increased the binding of 35S-heparin to FN, whereas A2 and A9 did not affect it. mAbs A2, A9 and A32 showed good binding to HBF, the 40 kDa proteolytic fragment of human FN which contains both Hep H and IIICS (type III connecting segment). These mAbs inhibited B16 cell adhesion to the HBF (heparin binding fragment) by 3050%, the greatest inhibition being shown by mAb A32. Two synthetic peptides from the HBF, CS1 (peptide 1) from the IIICS region and peptide I from the Hep H region, also inhibited B16 cell adhesion to HBF by approximately 70 and 30%, respectively. These results suggest that maximal cell adhesion to the HBF involves both CS1 and Hep H. The inhibitory effects of the two peptides were linearly additive in combination, whereas the inhibitory mAbs A2, A9 and A32 showed synergistic additive effects with each of the peptides. This points to the existence of an additional important cell binding site in Hep H, other than peptide I. Recent independent evidence for an additional cell binding site in Hep H supports this view. Melanoma cellular receptor(s) for the Hep H region may be cell surface proteoglycans but do not appear to bind to areas of Hep H with high affinity for soluble heparin, as the latter was not an inhibitor of B16 cell adhesion to the HBF. The increased effectiveness of A32 in inhibiting cell adhesion, compared to A2 and A9, may be due to conformational effects which increase the binding of soluble heparin, but reduce affinity for the ceUular receptor. These results are discussed in context with other reports in the literature.

The initial attachment of cultured bovine corneal epithelial cells and stromal fibroblasts to two oxygen-containing synthetic polymers was studied. Cultured epithelial cells and stromal fibroblasts were seeded onto two oxygen-containing surfaces: 'tissue culture' polystyrene (TCPS) and a polymer film deposited by RF plasma deposition using a methylmethacrylate monomer (MMA/FEP). To establish the mechanism of cell attachment, the effect of the selective removal of the vitronectin and fibronectin from the serum used in the culture medium was tested. The attachment of cultured epithelial cells during the first 90 min of culture was reduced by 40% (TCPS)--80% (MMA/FEP) as a result of removing vitronectin from the medium. Attachment of these cells to TCPS was reduced by 85-95% when the serum was depleted of both fibronectin and vitronectin. However, depletion of fibronectin reduced cell attachment to TCPS by 20%, whilst on MMA/FEP cell attachment was equivalent, or higher, than that for intact serum. The attachment of cultured corneal stromal fibroblasts was similarly dependent on vitronectin but less dependent on fibronectin. Therefore, for the attachment of both cultured epithelial cells and fibroblasts to oxygen-containing surfaces in the presence of serum, there is a high requirement for serum vitronectin but a lesser requirement for fibronectin. The effects of the establishment of corneal epithelial cells in culture and the site of origin of the cells, were determined. Primary isolates of epithelial cells isolated from the limbal, central or peripheral regions of the cornea were less dependent on vitronectin for initial attachment to TCPS than were these cells after several passages in culture. Furthermore, the primary isolates were dramatically less responsive to vitronectin than the cultured cells. These results indicate that the mechanism of attachment of corneal epithelial cells to TCPS varies with the culture experience of the cells. Cells that are culture neophytes can employe endogenous mechanisms for the initial attachment to TCPS, whereas cells established in culture are dependent on exogenous vitronectin in order to attach.