Determination of platelet monoamine oxidase activity in human platelet-rich plasma--a new microfluorescent assay utilizing kynuramine as substrate.
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Biomedical subjects
Publications and source records attributed to B W Papermaster.
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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.
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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.
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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.
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A fraction with lymphokine properties was isolated from supernatant medium of the continuous cultured human lymphoblast cell line, 1788. Culture medium containing 2% human serum was used for cell growth in order to minimize antigenicity of supernatant fractions isolated from the medium. The culture medium was passed through an Amicon XM-100 membrane, concentrated over a PM-10 membrane, lyophilized, and reconstituted to a final concentration of approximately 40:1. Studies in vivo and in vitro showed that the active fraction contained skin reactive factor (when injected intradermally into guinea pigs and humans), lymphotoxin, migration inhibition factor, chemotactic factor, and macrophage activation factor. This same preparation, when injected intralesionally into cutaneous tumors, induced an inflammatory reaction followed by tumor regression. The fraction confined between membranes of pore size 10,000-100,000 daltons was active in promoting tumor regression, while the fraction less than 10,000 daltons was inactive. Patients with skin lesions from metastatic carcinoma of the breast and other malignancies were studied, and 16 out of 30 treated lesions were judged to have undergone either complete or greater than 50% regression. Of these, 8 were biopsied before and after lymphokine injection, and 6 out of 9 were negative for tumor cells. Additional studies in vitro with material fractionated on Sephadex G-200 indicated that the macrophage-activating component binds to alpha-2 macroglobulin in the culture medium.