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Biomedical subjects

R F Ashman

Publications and source records attributed to R F Ashman.

68 records · Page 4Linked to original sources
PubMed1978

A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.

A two-step centrifugation procedure has been developed to isolate greater quantities of highly purified sheep erythrocyte antigen-binding cells (ABC) than previously possible. The first step involves partially separating sheep erythrocyte rosettes from unrosetted lymphocytes by their difference in buoyant density on Ficoll-Hypaque. Subsequent passage through a linear 5 to 10% Ficoll gradient produces further purification of rosettes by sedimentation velocity. Approximately 4.5 X 10(6) ABC can be obtained at 50 to 100% purity from 10(9) immune spleen cells (5 days post-immunization) and 1 X 10(5) ABC at 20 to 40% purity from 10(9) nonimmune spleen cells. The purified ABC from 5-day immune animals are 80 to 90% B cells and 10 to 20% T cells, and represent between 30 and 40% of the original ABC in the spleen cell population. Less than 0.2% of the purified ABC are plaque-forming cells (PFC) and less than 2% have intracellular immunoglobulin (Ig) or J chain. The quantities of ABC obtained are sufficient for investigating biochemical parameters of antigen-induced lymphocyte activation and for direct analysis of the surface isotypes found on antigen-binding cells after immunization.

Animals↗
PubMed1977

Regulation of immunoglobulin production in human peripheral bood leukocytes: cellular interactions.

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This system was shown to be totally T lymphocyte dependent as purified B lymphocytes (less than or equal to 1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period. T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished. T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes campable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populations in the mouse.

Adult↗
PubMed1976

Enzymatic modification of lymphocyte receptors for antigen. III. Resistance of receptors to trypsin at the peak of the immune response.

Using sheep erythrocytes (SRBC) as the antigen, two subpopulations of spleen antigen-binding lymphocytes could be distinguished by a marked difference in the susceptibility of their receptors to trypsin. In unimmunized animals, 30% of the antigen-binding cells were trypsin-resistant, whereas at 5 days after immunization, 80-90% were trypsin-resistant, indicating an increase of about 50-fold in trypsin-resistant antigen-binding cells per spleen. In contrast, trypsin-sensitive cells per spleen were only 4-fold higher on day 5 than before immunization. The rise in % trypsin sensitivity preceded the increase in rosettes per spleen, implying that immunization produced a preferential increase in trypsin-resistant antigen binding cells partly by converting sensitive cells to resistant cells. After the 5th day, the trypsin sensitivity of antigen-binding cells slowly returned toward the unimmunized level, but a booster injection of SRBC restored trypsin resistance. Trypsin resistance was not lost in the presence of sodium azide or protein synthesis inhibitors. But a slightly increased trypsin susceptibility was conferred by 2-deoxyglucose, implying that glycolysis or the glycosylation of protein may be involved in maintaining trypsin resistance.

Animals↗
PubMed1975

Enzymatic modification of lymphocyte receptors for antigen III. loss of antigen-responsiveness with recovery after adoptive transfer.

Spleen cells from bovine serum albumin (BSA)-primed CBA mice were exposed to proteolysis by papain and pronase under conditions where cell recovery and viability were not impaired but surface Ig and antigen-binding were greatly reduced. The ability of the cells to respond to BSA was tested by a modified Farr assay of serum taken 10 days after adoptive transfer of cells to lethally irradiated syngeneic recipients. Cells not exposed to enzymes were able to respond to BSA either in vitro before adoptive transfer or in vivo after transfer. Cells exposed to papain and pronase were unable to respond in vitro, but normal antibody production was approached when the cells were exposed to antigen in vivo. Thus, after removal of much surface Ig the cells were temporarily unresponsive to antigen but were still functional, as demonstrated by their ability to recover responsiveness in vivo.

Animals↗
PubMed1973

Direct demonstration of theta-positive antigen-binding cells, with antigen-induced movement of thymus-dependent cell receptors.

Anti-thetaAKR antibody conjugated to fluorescein has been used in direct immunofluorescence tests to identify spleen theta(+) (T) sheep erythrocyte rosette-forming cells in AKR mice. Specificity studies involving A and cogenic A/thetaAKR mice clearly demonstrated that the cell surface fluorescence and cytotoxicity produced by the antiserum is directed solely toward the thetaAKR alloantigen. Approximately (3/8) of rosette-forming and non-rosette-forming spleen cells were found to be theta(+). The tendency for T cells to bind less antigen and the tendency for antigen-binding T cells to bear less theta than other spleen T cells, first suggested by other studies involving rosette-elimination by anti-thetaC3H plus complement, were confirmed by direct immunofluorescence. All AKR rosettes are specifically inhibitable by anti-immunoglobulin, including T rosettes. Antigen-induced redistribution of T cell receptors, analogous to that previously described for B cell receptors (16), occurs as readily in theta(+)RFC as in theta(-) RFC, without altering the symmetrical ring distribution of thetaAKR antigen.

Animals↗