In searching for a suitable animal model system to study colorectal tumor-cell metastasis, the method of injecting tumor cells directly into the spleen of athymic nude mice was explored. The cells used in this study consisted of the Colo 205 cell line, isolated and established from the ascitic fluid of a patient with adenocarcinoma of the colon. In spite of showing tumorigenicity in nude mice after subcutaneous injections, anchorage-independent clonal growth in soft agar, and invasion in the chick embryo skin, the Colo 205 cells failed to metastasize to the lung or liver after intrasplenic injections. The effects of interferon, and interferon in combination with 5-fluorouracil (5-FU), on tumor formation in the spleen, were studied. The combined interferon- and 5-FU-treated animals displayed a significant difference from the controls in the generation of spleen tumors. This combination may possess potentially useful applications in the treatment of solid tumors such as those in the colon and rectum.
A method for determination of total tissue flavins (riboflavin, FMN, and FAD) involves extraction with 0.1 N HCI and microbiological assay of the extract using riboflavin as the standard ( 1 ) . The growth response of L a d obacillus casei (ATCC 7469) to these flavin compounds, based on acid production, has been noted to be identical(2).We present evidence here that shows that the growth response of this organism, measured turbidimetrically, differs significantly between the 3 flavin compounds, riboflavin, FMN, and FAD.Freshly prepared standard solutions of riboflavin, FMN, and FAD (Sigma. Chemical Co., St. Louis, Mo.) (100 ppmol/ml) were used for each assay. Ten separate assays were conducted. Six assays covered the range from 0 to 50 Fpmole per tube in 10 ppmole increments and 4 assays covered the range from 0 to 60 ppmole per tube in 20 ppmole increments.An aliquot of the standard solution was placed in an assay tube and sufficient water was added to give a volume of 1.5 ml. Doable strength assay media (Difco Laboratories, Detroit, Mi&) was then added to a final volume of 3 ml. All 3 compopunds were assayed concurrently in triplicate. The assay tubes were sterilized by autoclaving, cooled, and inoculated with a freshly prepared inoculum of L. casei (ATCC 7469) (3) a After incubation at 37°C for a period ranging from 21-22 hours, growth was measured turbidimetrically a t 650 qp. The results are expressed in terms of absmbancy units/ppmole flavin.Paper chromatographic analysis of the standard compounds was conducted in 3 of the experiments using the solvent systems described by Huennekens and Felton (4). Statistical analysis was by the "t" test as described by Snedecor ( 5). -* Abbreviations : FMN = flavin mononucleotide, FAD = flavin adenine dinuckotide. 0.2 '650MP 0. I 1 0 20 30 40 50 60 /L/LMOLES FLAVlN /ASSAY TUBE FIG. 1.The results (Fig. 1) show that there is a significant difference between riboflavin and FMN and a highly significant difference between ribo'flavin and FAD and between FMN and FAD with regard to the growth response of L. casei. A linear respnse of L. casei to all flavins tested was noted in all experiments.In the acid extraction of tissue Aavins there is a degradation of the FAD to FMN but little or nu breakdown of the FMN to riboflavin(6-8). Riboflavin per se accounts for a quantitatively insignificant amount of the total tissue flavin (8). The results presented here clearly show that the growth response to riboflavin and FMN is not the same when measured as described. The use, therefore, of riboflavin as a standard in the microbiological assay for tissue flavin extracted by acid would lead to quantitatively inaccurate results because very little of the extracted flavin is actually ribolflavin.
Sheep red blood cells (SRBC), when incubated with human peripheral blood lymphocytes (PBL),2 form rosettes (1). The lymphocytes that participate in rosette formation by adhering to SRBC are generally believed to be T lymphocytes (2). These rosettes have been shown to disintegrate rapidly if incubation of the reaction is carried out at 37°C (3). In striking contrast, however, is the behavior of rosettes formed by human thymus cells, which are highly stable and do not disintegrate even after prolonged incubation at 37°C (4). Galili and Schlesinger (5) reported that human PBL, when treated by allogeneic stimuli in mixed lymphocyte cultures, were resistant to the degradative effects of 37°C incubation on the rosettes formed with SRBC. They suggested that the T lymphocytes were activated in mixed lymphocyte cultures to form stable E-rosettes.
The role of macrophages in the transformation of human lymphocytes by the mitogen galactose oxidase was studied. Monocyte depleted, purified T or B cells did not undergo blastogenesis after treatment with galactose oxidase. When galactose oxidase-treated purified T cells were cultured with macrophages, a slight proliferative response was obtained. B cells treated similarly showed no response. When macrophages were treated with galactose oxidase, and then T & B, T, or B cells were added, proliferative responses were observed in all 3 categories. Finally, supernatants of media in which galactose oxidase-treated macrophages were cultured also demonstrated the property of stimulating lymphocyte transformation. These results are consistent with current concepts of mitogen "presentation" and the elaboration of soluble factors by macrophages in mediating the activation of lymphocytes in response to stimulating agents.
Following on an earlier study which demonstrated that PHA can activate human lymphocytes to form temperature-stable (37 degrees C.) E-rosettes with sheep red blood cells, a number of other mitogens were tested. These included Concanavalin A, pokeweed mitogen, soybean agglutinin, sodium periodate, and galactose oxidase. All except soybean agglutinin demonstrated the capability to activate lymphocytes to form stable E-rosettes.
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