Ben W. Papermaster

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.

Association of macrophage-activation factor from a human cultured lymphoid cell line with albumin and α2-macroglobulin

Macrophage-activating factors (MAF) were partially purified from the supernatant culture medium of the long-term human lymphoid cell line RPMI 1788 by gel filtration on Sephadex G-200 and preparative acrylamide slab-gel electrophoresis. The culture medium contained 2% human serum instead of the usual fetal calf serum. Macrophage activation properties were associated with α2-macroglobulin and albumin from the cell culture medium, and α2-macroglobulin was the more active on a mole for mole basis. Two assays utilizing in vivo and in vitro activation were used: (i) cytotoxicity in an adherent population of DBA2 mouse peritoneal cells against the syngeneic L1210 lymphoma and (ii) measurement of acid phosphatase in adherent cells. Since α2-macroglobulin and albumin obtained from control, nonincubated medium containing 2% human serum were inactive, it was suggested that α2-macroglobulin and albumin act as carrier molecules for a smaller molecular weight factor(s), which is the true active moiety.

Lymphokine‐mediated immunotherapy studies in mouse tumor systems

Studies have been performed using two subcutaneously implanted mouse tumor models to investigate the immunotherapeutic potential of lymphokine‐containing culture supernatants from long‐term human lymphoblast cell cultures. Human lymphoblastoid cell line, RPMI 1788, was used as a cell culture source of lymphokines. Supernatants were removed from cultures at the stationary phase of growth and concentrated on Amicon filters retaining molecules above 10,000 Daltons. This concentrate was applied to a Sephadex G‐25 column, equilibrated with ammonium bicarbonate buffer, for removal of salts and dye from the culture medium. The effluent was lyophilized and reconstituted for use in further purification by affinity chromatography and SDS‐PAGE gels. Such preparations were used to inject DBA/2 mice bearing subcutaneous L‐1210 tumors. In addition, the B‐16 melanoma was used as a model of a solid tumor in C57BI/6 mice. Animals were treated intralesionally and intraperitoneally with lymphokines containing preparations and control solutions. Tumors growing subcutaneously were susceptible to lymphokine‐induced inflammation‐mediated regression without additional therapy. In the study of L‐1210 subcutaneous tumors, reduction in tumor size was followed by complete regression, prolonged survival, immunity to additional inoculation, and cures in 20–40% of the treated mice. Tumor regression and prolongation of survival were also noted in mice bearing B‐16 melanomas. These studies support the use of mouse tumors as bioassays for antitumor inflammatory activity of human lymphokine preparations and help to quantitate their potential use in human tumor immunotherapy.

A new assay for rapid measurement of MIF levels by 3H-labelled cells in liquid scintillation counting vials: Statistical implications for the measurement of migration inhibition

A new quantitative assay for migration inhibitory factor (MIF) employs 3H-labelled cultured mouse or human lymphoid cells migrating from capillary tubes. Capillaries filled with labelled cells are placed in liquid scintillation counting vials, along with the MIF-containing sample and are removed at the end of a five-hour incubation period. The residual, labelled cells which have migrated out of the tubes are solubilized and counted in a liquid scintillation counter. While cultured lymphoblast cells are routinely used in the assay, the method was checked against mouse and guinea pig peritoneal exudate cells in both the labelled cell technique and the conventional chamber assay. The assay is technically simple to perform and a useful tool for laboratory research purposes because of the short span of time needed to obtain the results. These advantages indicate a potential for automation and use of this assay in a clinical immunology laboratory. Statistical analysis of data from both assays demonstrated that the relative variation among replicates is lower in the labelled cell assay. The new assay also measured a significant difference between controls and MIF-containing samples when the migration index (MI) was greater than 80%. Criteria for significant inhibition of migration are discussed in regard to the use of analysis of variance (ANOVA) and other statistical procedures, and the inadequacy of a single measure, such as the MI, is discussed.

A rapid quantitative assay for lymphotoxin.

A rapid quantitative assay for lymphotoxin was developed with the use of a mouse cultured lymphoid cell line, L1210 as the target cell. The assay produces results which are substantially in agreement with assays employing fibroblasts as targets. After incubation with lymphotoxin containing samples, target cells were labelled with [3H]thymidine and harvested with a Multiple Automated Sample Harvester (MASH). The MASH allows multiple replicates to be obtained from which the calculation of an I50 (50% inhibition) point and lymphotoxin specific activities can be performed with high statistical reliability by means of probit transformation and analysis.

The use of a leukocyte cell line culture supernatant for skin reaction testing in malignant melanoma

A study was conducted to determine some of the potential applications of a human leukocyte culture supernatant or “lymphokine” preparation in cancer patients. The application evaluated in this study was the use of this preparation as a skin test reagent for evaluation of the inflammatory response following intradermal injection. The preparation was derived from the supernatant of a long‐term cultured lymphoblastoid cell line with migration inhibition factor (MIF) and other lymphokine activities. Dose response, histology and toxicity studies were done in 53 patients with malignant melanoma stage IIIB and IV. A dose response curve was observed for both erythema and induration at 12 and 24 hours, but not at 48 hours. An optimal intradermal dose for eliciting inflammation was determined and found to be five units. Histopathological evaluation of biopsy specimens showed a mixed cell reaction including granulocytes, eosinophils, lymphocytes and monocytes differing in lymphocyte content from the classical delayed type hypersensitivity (DTH) reaction in man. Compared with the response to recall antigens, only a weak correlation with the DTH response to the recall antigens was found. Our results support the conclusion that lymphokines may be used in the future to evaluate the ability to develop nonspecific inflammation in cancer patients, and that this inflammatory response can be obtained in a number of patients no longer capable of responding to recall antigens.

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).