Search PubMed⌕ Search

Biomedical subjects

C C Stewart

Publications and source records attributed to C C Stewart.

At least 109 records · Page 6Linked to original sources
PubMed1985

Macrophage-induced cytostasis: kinetic analysis of bromodeoxyuridine-pulsed cells.

The effect of tumoricidal macrophages on the cell cycle progression of six different cell lines was studied using an anti-bromodeoxyuridine (BrdUrd) monoclonal antibody to follow the traverse of BrdUrd-labeled cells. Exponentially growing cultured mammalian cells, from six different cell lines, were prepulsed with BrdUrd before exposure to tumoricidal macrophages. The cultured cells were then analyzed as a function of time for DNA content (by propidium iodide staining) and for BrdUrd incorporation (using a fluoresceinisothiocyanate [FITC]-conjugated anti-BrdUrd monoclonal antibody). The position of the cells in cycle and the progression of the BrdUrd-labeled cohort was followed using flow cytometry. The cell lines examined were: Colon 26, BALB/c-3T3, ST3T3 (a spontaneously transformed, tumorigenic clone of 3T3), WCHE5 (a clone of whole Chinese hamster embryo cells), RIF (a radiation-induced fibrosarcoma), and A101D (a human melanoma). The bivariate distributions showed that for all six cell lines the BrdUrd-labeled cohort in the control cultures progressed around the cell cycle during the first 12 h of culture, as the cells exponentially increased. In contrast, when each cell line was incubated with tumoricidal macrophages, the BrdUrd-labeled cohort did not progress through cell cycle but remained in S phase throughout the 12-h culture period. There was also no evidence for progression of cells out of G1. The data show that cells were arrested in every phase of cell cycle. This study suggests that cytostasis, as manifested by the termination of progression in all phases of the cell cycle, is a universal phenomenon induced by tumoricidal macrophages.

Animals↗
PubMed1985

Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis.

Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogeneous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2Kk) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and gamma 2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages.

Animals↗
PubMed1985

A gelatin sponge model for studying tumor growth: flow cytometric analysis and quantitation of leukocytes and tumor cells in the EMT6 mouse tumor.

This study examined the recruitment of host cells into a progressing EMT6 tumor following the inoculation of tumorigenic cells into a preimplanted gelatin sponge. Tumor cell proliferation occurred to a greater extent in sponge tumors than in tumors obtained by direct subcutaneous injection of tumor cells. Blank sponges, lacking tumorigenic cells and used as controls, elicited an inflammatory response characterized by a modest infiltration of granulocytes and macrophages whose numbers, after Day 7 postimplantation, remained unchanged for 21 days of sponge residence in the animal. In contrast, when the sponge contained tumor cells, there was a continuous increase in host cell infiltration that paralleled the increase in tumor cells. Whether in a sponge or not, tumor cells represented more than half of the recovered cells by Day 21 after tumor cell inoculation. Macrophages comprised the greatest fraction of host cells infiltrating the tumors. Flow cytometric analysis and morphological examination of disaggregated tumors indicated that macrophages (19-51%) made up the largest proportion of host cells followed in order by granulocytes (6-18%) and lymphocytes (2-9%). Sorting of marker-labeled cells revealed that 94% of the surface immunoglobulin-bearing cells were macrophages. Twenty-two % of the cells bearing the Thy 1.2 marker were lymphocytes, and 68% were macrophages. The data confirm the occurrence of a cellular host response in the tumor-bearing animal which is distinct from the foreign body reaction elicited by a blank sponge. Additionally, the sponge system described here represents a recoverable environment that would facilitate the study of in vivo host-tumor cell interactions that occur during early tumor development or later during therapy-induced tumor rejection when a palpable tumor is not present.

Animals↗
PubMed1984

Cultivation of murine bone marrow macrophages in sponges: a method that permits recovery of viable cultured cells.

Various investigators have cultured murine bone marrow or peritoneal cells in vitro on glass or plastic surfaces with the ultimate aim of retrieving adherent macrophages for morphologic and functional evaluation. The removal of these adherent macrophages by conventional techniques has been consistently accompanied by low yield and significant cell damage. We report here a simple technique for culturing murine bone marrow cells in gelatin sponges (Spongostan and Gelfoam) in growth medium containing 10% fetal bovine serum and 10% L-cell conditioned medium. Viable cells were retrieved from the sponges in 10 min by digestion with collagenase. The in situ growth kinetics were similar to those found for cells cultured on plastic dishes. The recovered cells were adherent, phagocytic, positive for Fc gamma receptors, and had esterase activity.

Animals↗
PubMed1983

Cd2+ responses of cultured human blood cells.

Cd2+ cytotoxicity, uptake, and partitioning, and Cd2+-induced metallothioneine synthesis were studied in cultured peripheral human blood cells. Mononuclear cells were found to resist relatively high levels of Cd2+. Few cells were killed below 50 microM Cd2+. Above this value, survival decreased exponentially with dose. The mean LD50 for mononuclear cells cultured in Cd2+ for 40 hr was 100 microM. Polymorphonuclear cells (granulocytes) were found to be more resistant, with a significantly higher threshold and LD50, and a more complex dose response. Most of the Cd2+ incorporated by blood cells was taken up by nucleated cells. Despite their greater resistance, polymorphonuclear cells incorporated more Cd2+ at higher doses (50 to 150 microM) than did mononuclear cells. No Cd2+ was bound to metallothioneine in polymorphonuclear cells following exposure to Cd2+ for even extended periods of time (18 hr) at high doses of 109Cd2+ (25 microM). Instead Cd2+ appeared in a Sephadex G-75 peak of approximately 60,000 Da, as well as in the void peak. No significant amount of preexisting metallothioneine (MT) or metallothioneine mRNA was found in the mononuclear cells. However, MT synthesis was induced rapidly following exposure to Cd2+. [109Cd2+]MT appeared within 1 hr following exposure to 50 microM 109Cd2+, and MT synthesis rates measured from [35S]cysteine incorporation were found to be maximal within 4 hr.

Adult↗
PubMed1983

The effect of serum on human marrow mononuclear cell proliferation and maturation.

The effect of different serum sources on the growth of human bone marrow mononuclear cells in liquid culture was investigated. Newborn calf serum failed to support growth in liquid cultures whether or not exogenous colony-stimulating factor was present. Neither adherent nor nonadherent cells proliferated in medium supplemented with horse serum. Fetal calf serum allowed proliferation of the nonadherent cell population, but only in the presence of colony-stimulating factor. However, no growth of adherent cells was observed in these cultures. Both the nonadherent and adherent populations grew well in the presence of pooled human sera. While growth of the nonadherent population was minimal in the absence of colony-stimulating factor, the adherent population appeared to increase to a greater extent when the factor was absent. The positive effect of the human serum was shown to be dose dependent in that as the proportion of serum was increased in the medium, the number of cells recovered from the cultures increased, regardless of the presence or absence of exogenous colony-stimulating factor.

Blood↗
PubMed1983

Local proliferation of mononuclear phagocytes in tumors.

The fraction of macrophages and tumor cells in the DNA synthetic phase of the cell cycle was determined for the transplantable fibrosarcomas, RIF and KHT tumors, the mammary tumors EMT6, S102, and 67A, the B 16 melanoma, and the Lewis lung carcinoma. Thirty minutes after intraperitoneal injection of 3HTdR (tritiated thymidine), 10% to 28% of the macrophages were labeled. A similar proportion of tumor cells was labeled. This represents a high degree of local proliferation for macrophages in situ. Thus, macrophages can proliferate in growing tumors, and this local proliferation may represent an important source of these host defense cells.

Animals↗
PubMed1982

Phagocytosis: flow cytometric quantitation with fluorescent microspheres.

The phagocytosis of uniform fluorescent latex particles by pulmonary macrophages in the rat was analyzed by flow cytometric methods. The percentage of phagocytic macrophages and the number of particles per cell were determined from cell-size and fluorescence histograms. A comparison of in vivo and in vitro phagocytosis data showed that the percentage of phagocytic lavaged macrophages reflected the availability of instilled particles. With sodium azide used to model phagocytosis inhibition, it was shown that the percentage of phagocytic cells and the number of particles per cell can be determined simultaneously.

Animals↗
PubMed1982

Three-color fluorescence measurements on single cells excited at three laser wavelengths.

A three-laser flow cytometer for quantitative analysis and sorting of cells and microscopic particles has been developed and evaluated using argon- and krypton-ion lasers as excitation sources. Cells stained with three fluorescent dyes having different excitation spectra enter a flow chamber where they first pass through an electronic volume sensor and then intersect three spatially separated laser beams for fluorescence excitation of bound dyes and light scatter measurements. Separate pairs of beam-shaping optics independently position and focus each beam onto the cell stream thus permitting cells to be sequentially illuminated. Fluorescence is measured by a detector that employs a single collecting lens for single or multilaser excitation experiments. Fluorescence signals are processed and displayed as frequency distribution histograms using an LSI-11 computer. This instrument is described in detail with illustrative examples using cells stained for DNA content, protein, and cell surface antigens and cells that have phagocytized fluorescent microspheres.

Animals↗
PubMed1982

Quantitation of cell concentration using the flow cytometer.

A simple procedure has been developed to simultaneously measure cell concentration and analyze marker positive cell populations using the flow cytometer. Fluorescent latex particles of known concentration are added to the sample to be analyzed. Marker positive cells and fluorescent particles are then analyzed using flow cytometric methods. Since the particle concentration is known, the number of particles that have accumulated gives the exact volume of the sample analyzed. The concentration of total and marker positive cells that were analyzed then can be calculated. The method will be illustrated using single, dual, and triple laser excitation of cells stained for DNA content in combination with green or red fluorescent spheres. The usefulness of the procedure is demonstrated by counting cells from highly necrotic tumors where large amounts of debris are present.

Animals↗
PubMed1982

The activation of cloned macrophages by concanavalin A for tumoricidal effect: assessment of tumor cell cytotoxicity by a clonogenic assay.

The activation of pure populations of cloned peritoneal macrophages for tumor cell cytotoxicity by Con A was demonstrated using a recently developed tumor cell clonogenic assay. Cloned macrophages were rendered cytotoxic by Con A at concentrations above 20 micrograms/ml. The tumor cell cytotoxicity was caused mainly by the tumoricidal activity of the Con A-activated cloned macrophages. Increasing The Con A-activation time from 24 hours to 48 hours and 72 hours heightened the cytotoxic activity of cloned macrophages. Cloned macrophages incubated with con A for only 2 hours possessed no cytotoxic effect. Culture fluid from cultures of activated macrophages exerted no tumor cell cytotoxicity. Alpha-methyl-D-mannoside, a specific receptor-binding inhibitor for Con A, was capable of blocking the activation of macrophages for cytotoxicity at 0.01 M concentration.

Adenocarcinoma↗
PubMed1982

Effects of serum fractions on the growth of mononuclear phagocytes.

The effects of serum fractions on the growth kinetics and colony formation of mononuclear phagocytes derived from mouse bone marrow, blood, and peritoneal cavity were investigated. Peritoneal exudate macrophages and blood monocytes required a factor(s) found to reside in the nondialyzable serum fraction (molecular weight greater than 12,000) to survive, a small molecular weight (less than 307) factor(s) with growth-stimulatory activity (GSA) contained in the dialyzable serum fraction, and the macrophage growth factor (MGF) for proliferation and colony formation. Fetuin, a major protein of fetal serum, was able to substitute the nondialyzable serum fraction. Macrophages cultured in medium containing MGF and the nondialyzable serum fraction for 6 days could be restored to full growth following the addition of the dialyzable serum fraction. In contrast, bone marrow mononuclear phagocytes cultured in the absence of the dialyzable serum fraction were capable of proliferating, though at a slower rate, and forming colonies. In addition, neither insulin nor hydrocortisone was capable of replacing the serum-dialyzable GSA nor able to enhance colony formation.

Animals↗
PubMed1982

Growth regulation by macrophages.

The evidence reviewed here indicates that macrophages, either acting alone or in concert with other cells, influence the proliferation of multiple types of cells. Most of the data indicate that these effects are mediated by soluble macrophage-elaborated products (probably proteins) although the role of direct cell-to-cell contacts cannot be ruled out in all cases. A degree of success has been achieved on the biochemical characterization of these factors, but such work has been hampered by the factors low specific activity in conditioned medium and the lack of rapid, specific assays. It is our belief that understanding the growth-regulating potential of macrophages is an important and needed area of research.

Animals↗
PubMed1982

Role of the small bowel in regulating food intake in rats.

This study tested the hypothesis that exposure of the lower intestine to nutrients decreases subsequent food intake. Eight male Sprague-Dawley rats underwent jejunoileal bypass surgery with 8 cm of proximal jejunum anastomosed to 5 cm of terminal ileum in an end-to-side fashion. Eight control rats had sham bypass surgery with similar intestinal incisions that were reanastomosed in normal continuity. Another group of eight rats underwent ileal transposition (IT). A 10-cm segment of terminal ileum was transected, leaving the mesentery and blood supply intact, and transposed to the jejunum 8 cm from the ligament of Treitz. Thus total intestinal length was intact. Eight control rats had similar intestinal transections reanastomosed in normal continuity. A similar pattern of decreased food intake in the first 1-2 wk occurred in both bypass and IT rats. The values were significantly different from the respective sham surgery control groups. Body weights decreased, but the fall was greater in bypass rats. These data suggest that partially digested food in the lower intestine decreases food intake.

Animals↗
PubMed1981

Macrophage growth inhibitors derived from the murine peritoneal cavity.

The murine peritoneal cavity contains factors that inhibit the in vitro growth and colony formation of macrophages. The inhibition of macrophage growth is not due to cell death. In the presence of inhibitors, the growth of colony-forming macrophages is suppressed, and small clusters are formed as a result of limited proliferation. The more mature mononuclear phagocytes (blood monocytes and peritoneal exudate macrophages) are more sensitive to the overall inhibitory effect of the peritoneal inhibitors than the less mature bone marrow mononuclear phagocytes. Furthermore, using dialysis and Amicon ultrafiltration, at least two inhibitors with differential inhibitory effects can be demonstrated. The colony formation of bone marrow mononuclear phagocytes is suppressed mainly by a protease-resistant, small molecular weight (less than 1,000) dialyzable inhibitor. In contrast, peritoneal exudate macrophages are sensitive to both the small molecular weight inhibitor and a protease-sensitive, large molecular weight (greater than 12,000), nondialyzable inhibitor. The data suggest a possible existence of a dual inhibitor control on the proliferation of mononuclear phagocytes in vivo. In addition, the in vitro cultured peritoneal exudate cells are capable of producing inhibitors that mimic the activity of the in vivo inhibitors.

Animals↗