Purification and characterization of a novel human angiogenic factor (h-AF)

Serum-free supernatants of the human melanoma cell line A-375/2 contain an angiogenic activity as detected by the chorioallantois membrane assay which does not induce proliferation of cultured endothelial cells. This human angiogenic factor (h-AF) was purified by immunoaffinity chromatography as well as by conventional chromatographic procedures. A monoclonal antibody designated 5F4 was raised against h-AF, which binds but does not neutralize angiogenic activity. h-AF consists of a protein at MW 67 kD which focuses at pH 5.0. By biological and biochemical criteria it is shown that h-AF differs from other known growth factors.     

The biochemistry and in vitro activity of soluble factors of activated lymphocytes

Summary Activated lymphocytes release numerous products which are either synthesized de novo or in increased amounts; some of these products play a role in the regulation of the immune response and are designated as mediators of cellular immune reactions or lymphokines. The first lymphokine described was the macrophage migration inhibitory factor (MIF) which has been studied most extensively with regard to its chemical and biological properties. Using sensitive radiolabelling techniques and an antiserum against highly purified fractions of MIF we were able to identify several products of activated guinea pig lymphocytes with different molecular weights of 15.000, 30.000, 45.000, 60.000 which all had an isoelectric point of 5.2 and were all inhibitory to macrophage migration. It is suggested, that these molecules are oligomers of a common subunit of molecular weight 15.000. It was further shown, that molecules of the same physical-chemical and serological characteristics are produced by activated B-cells, L₂C leukemia cells and growing fibroblasts, thus further substantiating earlier reports on the production of MIF by lymphoid and non-lymphoid cells. The described molecules were also shown not to contain determinants of the major histocompatibility complex and to be distinct from lymphotoxin, another lymphocyte activation product. It is concluded, that MIF is not sa single molecule but rather a system of structurally related molecules. Thier interaction with macrophages and possible relationship to macrophage activating factor is discussed.