J. Philip McCoy

Laminin supports neurite outgrowth from explants of axotomized adult rat retinal neurons.

The influence of laminin on neurite outgrowth from explants of adult rat retina and its distribution in normal and lesioned rat optic nerves were examined. Neurite outgrowth required the presence of laminin in the substratum, and as with a goldfish retinal explant system, was markedly stimulated by prior axotomy. Except for blood vessels and the nerve sheath, normal rat optic nerve was devoid of laminin immunoreactivity. Unlike results seen in the goldfish optic nerve, injury to the rat optic nerve induced no observable increase in laminin content or change in its distribution. The differences in the in vivo regenerative capacities of these two species may in part be related to the differences in their abilities to provide a proper substratum for axon regrowth.

Enzyme-linked lectin assay (ELLA): Use of alkaline phosphatase-conjugated Griffonia simplicifolia B4 isolectin for the detection of α-d-galactopyranosyl end groups

Alkaline phosphatase has been coupled to Griffonia simplicifolia I B4 isolectin using a one-step glutaraldehyde conjugation procedure. This enzyme-lectin conjugate (AP-GS I-B4) has been used to specifically detect plastic-bound natural and synthetic glycoproteins bearing alpha-D-galactopyranosyl end groups. The extent of reactivity of the AP-GS I-B4 with the glycoproteins appears to be proportional to the number of terminal galactosyl residues present. Furthermore, this assay, termed ELLA (enzyme-linked lectin assay), is specifically inhibitable by low-molecular-weight sugars containing terminal alpha-D-galactosyl groups. The ELLA reactions may be assayed rapidly and objectively by the use of commercially available ELISA-plate using standard filters.

Binding of Griffonia simplicifolia I lectin to rat pulmonary alveolar macrophages and its use in purifying type II alveolar epithelial cells

We report that the isolectin Griffonia simplicifolia I-B4 isolated from G. simplicifolia seeds binds to rat alveolar macrophages present in frozen sections of lung tissue or bronchoalveolar lavage fluid. G. simplicifolia I-B4 does not bind to alveolar epithelial cells. We established that G. simplicifolia I-B4 binds to the macrophages via interaction with terminal alpha-D-galactopyranosyl residues present on these cells. This was substantiated by demonstrating that binding is inhibited either by the haptenic sugar alpha-D-galactopyranoside or by treating the cells with coffee bean alpha-galactosidase. Because murine laminin is known to contain terminal alpha-D-galactopyranosyl end-groups, and because we found that an anti-laminin antiserum binds to rat alveolar macrophages, we suspect that G. simplicifolia I-B4 may be binding to laminin present on the macrophages. To isolate alveolar type II epithelial cells from rat lungs, we developed a method that utilizes the lectin G. simplicifolia I. When proteinase-derived suspensions of pulmonary cells are incubated with G. simplicifolia I, the macrophages agglutinate and can be removed by filtration through nylon mesh. After incubating the resulting cellular suspension in tissue culture, the adherent cells are 94 +/- 2% (S.D.) type II cells. When compared to cells isolated by repeated differential adherence, the lectin-prepared type II cells have similar morphology and staining characteristics, form domes in monolayers and incorporate similar amounts of palmitate into disaturated phosphatidylcholine. We believe that the procedure outlined in this report provides a simple and effective method to isolate type II alveolar epithelial cells from rat lungs.

Contribution of α-d-galactopyranosyl end groups to attachment of highly and low metastatic murine fibrosarcoma cells to various substrates☆

There are much greater numbers of cell surface terminal, non-reducing alpha-D-galactorpyranosyl groups in highly malignant (metastatic) cells than are found in low malignant cells derived from the same murine fibrosarcoma. We have examined the contribution of these residues to attachment of the cells to various collagens and to plastic. Removal of these carbohydrate groups with alpha-galactosidase or blocking them with lectins from Griffonia simplicifolia seeds or with anti-blood group B antiserum all dramatically inhibited the attachment of both the highly malignant and the low malignant cells. Following removal with the enzyme, the alpha-D-galactopyranosyl end groups were rapidly resynthesized. This resynthesis was inhibited by tunicamycin, an inhibitor of de novo glycoprotein synthesis. This antibiotic also impaired cell attachment and, when used in addition to treatment with alpha-galactosidase, it inhibited cell attachment more than did treatment with the enzyme alone. The effects of all treatments on cell attachment were greater for the highly malignant than for the low malignant cells. With the latter cells, inhibition by lectin was seen only in the absence of serum, whereas the adhesion of highly malignant cells was affected in both the presence and the absence of serum. On their surface membrane the highly malignant cells express much more than do the low malignant cells of a glycoprotein that cross-reacts immunologically with laminin. The basement membrane glycoprotein laminin promotes cell attachment to collagen, and both glycoproteins contain terminal, non-reducing alpha-D-galactopyranosyl groups. Attachment of cells is a requirement for the formation of a metastasis, and thus the laminin-like molecule and the alpha-D-galactopyranosyl end groups (whether on the laminin-related moiety or on other cell surface molecules) may both be important for expression of the most malignant phenotype.

Enzyme-linked Lectin Assay (ELLA) II. Detection of Carbohydrate Groups on the Surface of Unfixed Cells

An enzyme-linked lectin assay (ELLA) has been developed to detect specific carbohydrate units on the surface of unfixed cells. The assay may be read in standard ELISA plate readers, since the cell-bound enzyme-lectin conjugate is specifically eluted from the cells prior to development of the conjugate. ELLA, when read in an enzyme-linked immunosorbent assay (ELISA) plate reader, allows better detection and relative quantitation of specific surface carbohydrate units than is possible by standard immunofluorescence with fluorescein-conjugated lectins.

Attachment, spreading and growth in vitro of highly malignant and low malignant murine fibrosarcoma cells

Highly malignant cell lines and low-malignant cell lines isolated from three different methylcholanthrene-induced murine fibrosarcomas were examined for their ability to attach to plastic dishes and collagen-coated dishes under serumfree conditions and in the presence of serum. Most of the cells from the three highly malignant lines attached and spread under all conditions. By 72h, there was a significant increase in the number of cells indicating that at least some of the cells had undergone division (even in the absence of serum). In contrast, fewer of the cells from the three low-malignant lines attached and spread on the plastic or collagen substrates in the absence of serum or in the presence of 0.1 per cent serum. However, when 15μg laminin per dish was added along with the lowmalignant cells, they then attached and spread on the plastic and collagen-coated dishes. Previous studies have indicated that the highly malignant lines express cell surface antigens that cross-react with laminin while the low-malignant cell lines do not. We speculate that the differences between the high- and low-malignant cells in the expression of cell surface laminin-like antigens contribute to the dissimilarities in attachment and spreading capacity. These differences may also contribute to the dissimilarity between these cells in malignant potential.

Nuclear Parameters as Prognostic Indicators in Glioblastoma Patients

The objective of this study was to determine whether nuclear parameters were associated with prognosis in glioblastoma multiforme. DNA indices, cell cycle parameters, and nuclear population densities were compared with patient survival. Selection criteria included the pathologic diagnosis of a cerebral glioblastoma multiforme, absence of therapy before surgery, and adequate tissue to measure each nuclear parameter studied. Nine cases accrued over a two year period. The amount of DNA per nucleus was quantified in fresh tissue specimens by nuclear-isolation flow cytometry in Vindelovs solution. One particular association was significant when tested by Cox models: The percentage of nuclei with S-phase amounts of nuclear DNA was a significant predictor of decreased survival (regression coefficient=0.20, p=0.04). The percentage of nuclei in the Go/G, peak was marginally associated with longer survival. These data are evidence of an association between nuclei in certain phases of the DNA cell cycle and postoperative survival.

Connective tissue diseases and bovine collagen implants

Bovine collagen implants are biomaterial used for the correction of dermal contour deformities. The use of bovine collagen implants in patients with a personal history of autoimmune diseases is contraindicated by the manufacturer. In our study, sera from fifty patients with scleroderma, rheumatoid arthritis, and systemic lupus erythematosus were examined for antibodies to bovine collagen implants by means of a previously described enzyme-linked immunosorbent assay. None of the patients had received bovine collagen implants. The anti-bovine collagen implant antibody levels in the sera of patients with scleroderma, systemic lupus erythematosus, and rheumatoid arthritis were not, in general, statistically different from those in the normal population.

Cervical carcinoma antigen: Distribution in neoplastic lesions of the uterine cervix and comparison to other tumor markers

Abstract Four antibodies (anti-CCA, anti-CEA, Ca-1, and anti-EMA) were used to study the distribution of antibody-binding sites in normal endocervical mucosa, metaplastic squamous epithelium, squamous epithelium exhibiting varying grades of intraepithelial neoplasia, and invasive squamous cell carcinoma. Anti-CCA, a novel monoclonal antibody raised against an extract of squamous cell carcinoma of the cervix, recognizes dysplastic, neoplastic, and metaplastic cervical epithelial cells. While anti-CCA and Ca-1 rarely stained normal glandular epithelium, 31.4 and 45.7% of the samples stained positively for CEA and EMA, respectively. There did not appear to be significant differences between anti-CCA and the other antibodies in the frequency with which neoplastic conditions were stained. Based upon these observations, it appears that none of the antibodies tested can be regarded as a specific tumor marker.

The Attraction of Wandering Metastatic Cells

The cascade of events leading to the formation of metastases from primary tumors is a complex and incompletely understood phenomenon. The current concept envisions the initial invasion of local tissue and separation of individual cells or small groups of cells from the primary tumor. The invading cells gain access to the blood vascular and/or lymphatic circulatory systems and are distributed throughout the body. Most of the cells which enter the circulation, however, are rapidly killed. Only a few go on to establish themselves at secondary sites and carry on the processes that lead to the formation of metastatic growths. These processes include attachment to the appropriate structures (i.e., either the vascular endothelial cells or the sub-endothelial basement membranes), invasion of these structures and ultimately, proliferation. The process of tumor cell invasion through the endothelial layer and the sub-endothelial cell basement membrane most likely involves both active cell motility and substrate degradation.