John E. McEntire

Lymphokine-stimulated macrophage phagocytosis of fluorescent microspheres: a rapid new assay

A highly sensitive and rapid in vitro macrophage phagocytosis assay is described for screening of lymphokine preparations with macrophage activating properties. Non-induced mouse peritoneal macrophages were incubated on glass slides for 60 min in the presence of fluorescent 2 micron latex beads and partially purified lymphokine fractions or media control. Lymphokine samples were prepared from culture supernatants of the B lymphoblastoid cell line RPMI 1788 by high performance liquid chromatography of a soluble trichloroacetic acid extract of concentrated culture supernatant. Phagocytosis was measured by direct counts of the percent phagocytic cells and the number of intracellular beads per 100 cells as seen by fluorescence microscopy. Phagocytic uptake in the presence of as little as 0.1 ng of active lymphokine could readily be determined qualitatively by correlation plots and quantitatively by appropriate statistical programs for data processing by computer. This technique provides a rapid, reproducible, screening assay for biological activity of macrophage activating lymphokines and other compounds affecting macrophage phagocytosis.

Purification of alpha-2 macroglobulin by immunoadsorbent chromatography

A method for preparation of alpha-2 macroglobulin (alpha2M) by immunoadsorbent chromatography utilizing rabbit--anti-human alpha2M-conjugated Sepharose 4B is described. This procedure offers a rapid, simple and inexpensive method for purification of alpha2M from as little as 2.0 ml of human serum. By multiple applications to the absorbent column, as much as 10 mg of purified alpha2M may be easily prepared in one day. This technique is especially useful for isolation of alpha2M from individual sera and for studies of cultured cell supernatants containing human alpha2M.

Chapter 12 Serum Nucleoside Chromatography for Classification of Lung Cancer Patients and Controls

Publisher Summary This chapter describes serum nucleoside chromatography for classification of lung cancer patients and controls. Modified ribonucleosides derived predominantly from transfer RNA and ribosomal RNA, are known to be excreted in abnormal amounts in the urine of cancer patients. The molecular mechanisms of elevated excretion are unclear. Extract of neoplastic tissues have aberrant tRNA methyltransferases, and it has been suggested that the high turnover of tRNA is because of the rapid degradation of aberrantly modified tRNAs. Experimental evidence indicates that methylation of tRNA occurs only after-synthesis of the intact macro-molecule. No kinases have been found that will re-incorporate these compounds into tRNA, and consequently, they are excreted following metabolic degradation of tRNA. A characteristic of small cell carcinoma (SCC) is its tendency toward early and distant metastases estimated to occur in 70% of patients at the time of diagnosis. This very typical behavior of SCC creates problems in accurately defining tumor burden, and in estimating extent of disease despite technical advances in modern radiological techniques for demonstrating the presence of tumor in organs or tissues. As a consequence, difficulties are frequently encountered not only at initial staging but in correctly quantifying response and assessing sites of recurrent or metastatic disease.

Phase I Trial of Intravenous Lymphokine MAF

Preparation of large enough quantities of purified, chemically characterized biological response modifiers (BRMs) has required years of preparative and analytic studies leading to isolation and characterization of biologically active, pure peptides. Prior to the presently limited use of such peptides, phase I studies were carried out to determine biological effects of more complex preparations in patients, as in the case of human leukocyte interferon (Strander et al., 1973) and thymosin fraction 5 (Costanzi et al., 1977). Complex lymphokine fractions have been used for inducing local skin inflammatory reactions (Papermaster et al., 1976) and to treat dermal tumor lesions by direct intralesional injection (Paradinas et al., 1982) and also have been administered intravenously by Dumonde et al. (1981) to patients with disseminated cancer at low to moderate doses [1–2 ml delivering a total of 70 mg protein (Organon preparation)] and roughly equivalent to our unitage of between 200 and 500 units (Paradinas et al., 1982).