Biomedical subjects

M A Cuchens

Publications and source records attributed to M A Cuchens.

At least 37 records · Page 2Linked to original sources

PubMed1986

Kinetics of membrane immunoglobulin capping on murine B lymphocytes. Effects of phospholipid fatty acid replacement.

Detailed analyses regarding the effects of temperature and phospholipid fatty acid replacement on the capping of membrane immunoglobulin (mIg) have been performed using a recently described flow cytometric procedure (Cuchens, M. A., and Buttke, T. M. (1984) Cytometry 5, 601-609). Purified murine B cells were incubated for 12-20 h in the presence of bovine serum albumin-complexed 80 microM stearic (18:0), oleic (cis-18:1), or linoleic (cis, cis-18:2) free fatty acids. Unmodified and free fatty acid-treated cells were stained with fluorescein-conjugated rabbit anti-mouse Ig and subjected to pulse-shape (width) analyses to follow the kinetics of mIg capping. In both unmodified and free fatty acid-treated cells, capping of mIg occurred at all temperatures between 17 and 37 degrees C, but the rate of cap formation was temperature dependent. Arrhenius plots of mIg capping were linear, with activation energies ranging from 14 to 23 kcal/mol depending on the saturated/unsaturated fatty acid ratio of B cell phospholipids. Ligand-induced redistribution of mIg thus appears to be sensitive to changes in membrane acyl chain composition.

Animals↗

PubMed1986

The role of the Peyer's patch in carcinogenesis. I. The adsorption from the gut and retention of 3-methylcholanthrene by Peyer's patches.

Radiotracer methods were used to determine the distribution of 3-methylcholanthrene (3-MC) within the lymphoid organs of rats following i.g. intubation, i.l. injection into the small intestine, i.v. injection or direct injection of the Peyer's patches with 3-[6-14C]methylcholanthrene (14C-MC). The data indicate that the gut-associated Peyer's patches and mesenteric lymph nodes were exposed to higher amounts of orally administered 14C-MC than any of the other lymphoid organs. Whereas the Peyer's patches exhibited the highest sp. act. for longer periods of time when low amounts of 14C-MC were administered, the sp. act. of the mesenteric lymph node were greater when rats were intubated with higher amounts of 14C-MC. Furthermore, the Peyer's patches were exposed to higher amounts of possible metabolites of 14C-MC. Injection of 14C-MC into the small intestinal lumen resulted in increased ratios of the Peyer's patch sp. act. to mesenteric lymph node sp. act., indicating that by-passing the stomach altered the distribution patterns. Data from rats injected i.v. with 14C-MC demonstrated that mesenteric lymph nodes but not Peyer's patches adsorbed and retained 14C-MC from the blood and indicated that the 14C-MC associated with Peyer's patches of i.g. treated rats was adsorbed from the gut rather than from the blood. Results obtained from rats which were exposed to 3-MC by directly injecting Peyer's patches with 14C-MC also indicated that the Peyer's patches were able to retain 3-MC once localized within this lymphoid organ, to metabolize the 3-MC and to possibly excrete the polycyclic aromatic hydrocarbon into the small intestine. Collectively the data indicate that Peyer's patches have an important role in the adsorption from the gut and subsequent retention of 3-MC and hence may be a likely target organ for lymphoid carcinogenesis following oral exposure to carcinogenic polycyclic aromatic hydrocarbons.

Adsorption↗

PubMed1986

The role of the Peyer's patch in carcinogenesis. II. 3-Methylcholanthrene-induced lymphoid malignancies in rat Peyer's patches.

To determine whether B or T lymphoid malignancies could be induced following the exposure of lymphoid cells within different lymphoid organs to a potential chemical carcinogen, 3-methylcholanthrene (3-MC) was directly injected into surgically exposed Peyer's patches (PP), mesenteric lymph nodes (MLN), axillary lymph nodes (ALN) or spleens (SP) of Copenhagen rats. A high incidence of lymphoproliferative disorders was observed within rats which received 3-MC injections into the PP, but not in MLN, ALN or SP injected groups of rats. In addition to the PP environment, the dose of 3-MC and exposure to pristane were important factors in the induction of T versus B cell disorders. Whereas the B cell diseases were observed in pristane-treated rats which also received PP injections of low doses (5 or 50 micrograms) of 3-MC, in animals receiving a higher dose (500 micrograms) a much higher incidence of T cell disorders was detected. The observed lymphoproliferative diseases were categorized as B lymphocytic leukemias, B lymphomas or thymic lymphomas on the basis of histological examination of the tissues, white blood cell numbers and differentials, and the morphological and phenotypic characteristics of cell isolates. Abnormal DNA staining characteristics, increased soft agar cloning frequencies and metastasis of the leukemia or tumor cells indicated that malignant cells were associated with the proliferative diseases. Collectively the data indicate that primary lymphoid malignancies of either B or T cell origins may be preferentially and reproducibly induced by localizing low or high doses respectively, of 3-MC within the PP of rats exposed to pristane. These results suggest that PP may have a possible role in the carcinogenesis of lymphocytes following the exposure to chemical carcinogens via the gastrointestinal tract.

Animals↗

PubMed1986

Temperature-mediated processes in teleost immunity: binding and mitogenic properties of concanavalin A with channel catfish lymphocytes.

Low (non-permissive) temperatures inhibit the proliferation responses of channel catfish T cells to stimulation with Con A. The study reported here was undertaken to ascertain if failure to bind Con A at non-permissive temperatures could explain the observed suppression at such temperatures. The influence of temperature on the binding of fluorescein-labelled succinyl Con A to channel catfish T and B cells was studied by cytofluorography. The results indicated that Con A bound equally well at both mitogenically permissive (22 degrees, 27 degrees and 32 degrees) and non-permissive (12 degrees and 17 degrees) temperatures. Hence, temperature effects on mitogen-binding to cell surface receptors cannot readily explain the observed suppression of channel catfish T-cell proliferative responses at non-permissive temperatures.

Animals↗

PubMed1985

A procedure for the isolation of highly purified populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood.

A sequential separation protocol is described which reproducibly yields biologically active populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood. The purification protocol employed T lymphocyte rosetting with unmodified sheep red blood cells, monocyte adherence to microexudate-coated flasks and anti-immunoglobulin panning of B cells. Population purity was determined by cytofluorographic light scatter analysis, expression of cell surface markers, esterase activity and mitogen responsiveness. The sequential protocol reproducibly yielded viable and functionally active cell populations of greater than 95% purity from a single starting population of mononuclear cells.

Antigens, Surface↗

PubMed1984

Effects of different protein supplements on mitogen responses of human peripheral blood lymphocytes.

We evaluated the effects of 6 supplements often used in human lymphocyte cultures, including fetal calf serum, autologous human serum, pooled human AB serum, hypogammaglobulinemic human serum, bovine serum albumin and human serum albumin. Lymphocyte proliferation of unstimulated and mitogen activated peripheral blood mononuclear cells was measured by [3H]thymidine incorporation. The response of cells stimulated with the T-cell mitogen phytohemagglutinin-P were significantly lower when cultured in bovine serum albumin supplemented media, but were otherwise not supplement dependent. In contrast, responses of cells stimulated with the B-cell mitogens Cowan I strain of S. aureus and antisera against the mu or delta chain of human immunoglobulin were significantly effected by supplement. Cultures containing fetal calf serum and bovine serum albumin had high background responses without a proportional rise in cellular proliferation when B-lymphocyte-specific mitogens were utilized. Autologous human serum and pooled human AB serum contained immunoglobulin which interacted with each of the B cell mitogens, thus limiting their usefulness as in vitro supplements. Cells grown in human serum albumin supplemented media had minimal background and high stimulated responses to B-cell mitogens. These results indicate that human serum albumin is an optimal supplement for in vitro human lymphocyte proliferative assays since it supports high stimulated cell responses with low background activity, is devoid of immunoglobulin and had minimal variability among lots.

Adult↗

PubMed1984

A kinetic study of membrane immunoglobulin capping by flow cytometry.

A flow cytometric procedure has been developed for performing kinetic studies on the capping of membrane immunoglobulin (mIg) on B lymphocytes. Mouse B cells were stained with fluorescein-conjugated antimouse-Ig antisera and subjected to pulse-shape (width, peak, and area) analyses prior to, during, and after ligand-induced redistribution of mIg. It was found that ring-stained, patched, and capped cells could be discriminated based on the width of the electronic signal curve generated as the cells passed through the laser beam. Additionally, endocytosis and or shedding of the cap could be correlated with a change in the area under the curve. Using these two parameters (width and area), the effects of temperature, cross-linking, and several pharmacological agents on the capping process were examined. Through the use of flow cytometry, the inhibitory effects of various perturbants could be localized to discrete stages of the capping process.

Animals↗

PubMed1984

Temperature-mediated processes in teleost immunity: differential effects of in vitro and in vivo temperatures on mitogenic responses of channel catfish lymphocytes.

The in vitro mitogenic responses of channel catfish peripheral blood leucocytes to ConA and LPS were differentially affected by both in vitro and in vivo temperatures. The magnitude of the response to LPS was relatively independent of both in vitro culture temperature and in vivo acclimation temperature. The magnitude of the response to ConA was suppressed at lower in vitro temperatures although this suppression could be reduced by lower in vivo acclimation temperatures. In vitro temperature-shift experiments indicated that channel catfish PBL could respond to ConA at a lower in vitro temperature if first stimulated with ConA at a higher in vitro temperature. The converse, however was not true in that channel catfish PBL did not respond at a higher in vitro temperature after an initial stimulation with ConA at a lower in vitro temperature. This latter failure to respond could not be attributed to the induction of a suppressor cell (or factor) by exposure to ConA at a lower temperature. These studies, when coupled with other available data on channel catfish PBL subpopulations, are interpreted as supporting the hypothesis that low temperature immunosuppression in fish may result from preferential inhibitory effects on T cells rather than B cells.

Animals↗

PubMed1984

Automated immunofluorescent speciation of oral bacteria using flow cytometry.

Mixtures containing two bacterial species were analyzed using flow cytometric techniques. Light scattering characteristics of Streptococcus mutans and Actinomyces viscosus show unique profiles for pure cultures. However, the light scatter analysis of a mixture containing these two species demonstrates overlapping near the origin. Thus, light scatter analysis was not sufficient to speciate bacteria with different morphologies. Labeling of samples with species-specific immunofluorescent antibodies permitted speciation of mixtures. As the percentage of the bacterium to which the antibody is directed increased in a two-component mixture, fluorescent flow cytometric analysis showed a corresponding increase in the percentage of cells displaying fluorescent labeling. These methods could permit the rapid identification of bacteria from oral sites without culturing.

Actinomyces↗

PubMed1984

Inhibition of lymphocyte proliferation by free fatty acids. II. Toxicity of stearic acid towards phytohaemagglutinin-activated T cells.

Studies were performed to further characterize the effects of saturated fatty acids on murine T lymphocyte proliferation. Flow cytometry was used to show that the inhibitory effects of stearic acid (18:0) on [3H]thymidine uptake can be correlated with changes in cellular DNA content. Additional studies using flow cytometry and fluorescein diacetate as a viability stain showed that exogenous 18:0 was toxic for phytohemagglutinin (PHA)-stimulated T cells, whereas the viability of unstimulated T cells was less affected by 18:0. The inhibitory effects of 18:0 on T cell proliferation were evident as early as 4 hr after fatty acid addition and after a 10-hr exposure, the effects of 18:0 could not be reversed by washing the cells or by adding oleic acid (18:1). It is proposed that the inhibitory effects of 18:0 are dependent upon PHA-induced changes in T cell lipid metabolism.

Animals↗

PubMed1984

Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro mitogenic responses of channel catfish leukocytes.

Cell separation and enrichment techniques were employed to isolate three distinct leukocyte subpopulations present in channel catfish (Ictalurus punctatus) peripheral blood. Surface immunoglobulin-positive (sIg+) and sIg- lymphocytes were separated by an indirect "panning" technique employing monoclonal antibodies reactive with channel catfish Ig. A third cell population composed of macrophages was isolated by adherence to baby hamster kidney cell microexudate-coated surfaces. Functional features of these three subpopulations were assessed by in vitro mitogenic responses to lipopolysaccharide (LPS) and concanavalin A (Con A). The results that were obtained indicated that the sIg+ cells responded only to LPS stimulation regardless of the presence or absence of macrophages. The sIg- subpopulation, however, responded to neither LPS nor Con A unless macrophages were present, in which case responses were obtained to both mitogens. The accessory cell nature of the macrophages was shown by experiments utilizing fixed numbers of one cell type mixed with varying numbers of another cell type. Furthermore, the accessory cell function was abrogated by passage through Sephadex G-10 and preincubation with L-leucine methyl ester. These studies provide further evidence that teleosts not only contain B and T cells akin to those in mammalian systems, but contain accessory cells (macrophages) as well.

Animals↗

PubMed1983

Positive selection of mouse B and T lymphocytes and analysis of isolated populations by flow cytometry.

Mouse spleen cells were separated into Ig+ and Ig- populations by positive selection using petri plates coated with rabbit anti-mouse immunoglobulin. The Ig- cells were subsequently incubated with mouse monoclonal alloantisera to Thy1.2 prior to a second positive selection. The adherent populations were characterized as B (Ig+) or T (Thy1.2+) lymphocytes on the basis of surface immunofluorescence and mitogen-induced proliferation. Analysis of the 2 isolated populations by flow cytometry showed that B and T lymphocytes could be distinguished by their forward light scatter as well as their fluorescence after incubation with fluorescein diacetate.

Animals↗

PubMed1982