A method previously used in this laboratory for entrapment of tumor cells in alginate beads has been extended to provide a slow release delivery system for growth factors with known in vivo angiogenic activity. Protein growth factors were entrapped in alginate beads in amounts sufficient to cause incorporation of 3H-thymidine by COMMA-D cells in vitro, and in vivo neovascularization when injected subcutaneously into Balb/c mice. Entrapment of 125I-labelled growth factors showed that the amount of molecule entrapped in alginate beads may vary with the charge of the molecule. In vitro cell proliferation studies showed that entrapment in alginate beads may provide a slow-release system or a stabilizing environment for the protein. In some cases biological activity of the growth factor in solution was increased by the presence of control alginate beads. When alginate-entrapped growth factors were injected into Balb/c mice, induction of new blood vessels could be monitored qualitatively by macroscopic photography and assessed quantitatively by measuring the pooling of radiolabelled red blood cells at the experimental site. Subcutaneous injection of purified angiogenic factors not entrapped in alginate beads did not cause neovascularization. Diffusion of 125I-labelled growth factors from alginate beads in the animal showed that release in vivo may depend on the charge of the protein molecule. These results indicate that injection of purified molecules entrapped in alginate beads provides an effective localized and slow-release delivery of biologically active molecules. This delivery system may extend the time of effectiveness of biologically active molecules in vivo compared to direct injection without alginate entrapment. The method of entrapment and injection has potential for identifying active factors in tumor-induced angiogenesis and testing new compounds as modulators of neovascularization.

Immunoenzyme staining for tissue antigens using glucose oxidase as marker enzyme was compared with immunoperoxidase techniques. Both the indirect, conjugated antibody method and the unlabeled antibody procedure employing preformed complexes of glucose oxidase-antiglucose oxidase (GAG) and peroxidase-antiperoxidase (PAP) were used to stain carcinoembryonic antigen (CEA) in paraffin tissue sections. The localization of CEA within a tissue specimen was the same in all cases, and background staining was minimal. The percentage of positive specimens detected with GAG and PAP was similar but was slightly greater with glucose oxidase compared to peroxidase conjugates. The glucose oxidase conjugates and GAG were similar to the comparable immunoperoxidase reagents in enzyme-antibody molar ratio, retention of antibody and enzyme activity, immunohistochemical staining dilutions, and stability. Immunoglucose oxidase and immunoperoxidase were combined to localize CEA and colon-specific antigen-p simultaneously. Excellent contrast and staining separation were shown between the enzymatic reaction products of the two systems. Since immunoglucose oxidase methods are as sensitive as the comparable immunoperoxidase techniques, they should be considered as a reliable alternative to the latter, especially when endogenous peroxidase activity may be a problem. Furthermore, the two methods can be conveniently combined for the simultaneous detection of two antigens.

The effect of soluble factors from the monocyte/macrophage (M0) on cell proliferation and the functional effects of cell-cell contact on the arachidonic acid (AA) cascade were studied with vascular smooth muscle cells (SMCs). Peripheral blood M0s were isolated by adherence or in a Percoll gradient, and alveolar M0s were obtained by lavage. Conditioned medium (CM) was prepared by preincubating M0s with medium alone or by separating SMC and M0 cocultures by a membrane insert Cell proliferation (image analysis) and 6-ketoprostaglandin F 1