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Biomedical subjects

R F Ashman

Publications and source records attributed to R F Ashman.

At least 37 records · Page 2Linked to original sources
PubMed1988

Membrane depolarization of human B cells follows stimulation by either anti-mu or B-cell growth factor, but only anti-mu causes cell volume changes.

Human peripheral blood B cells were separated from monocytes and T cells, depleted of null cells by an anti-Leu 9 rosetting technique, and fractionated on discontinuous Percoll gradients to yield a highly purified, small, dense B-cell population. These cells responded to F(ab')2 goat anti-mu at 10 and 100 micrograms/ml with membrane depolarization (measured by immunofluorescence with 3,3'-dipentyloxacarbocyanine dye) at 1 h, cell volume enlargement by 48 h, and modest thymidine incorporation by 72 h. They also responded to the 12-kd human B-cell growth factor of Maizel with membrane depolarization, but not with cell volume increase. F(ab')2 anti-mu and B-cell growth factor together induced greater depolarization than was seen with either alone, but there was no synergy. The cell volume increase seen with F(ab')2 anti-mu was not increased by B-cell growth factor. Comparison of data analysis methods showed that mean fluorescence intensity most readily detected significant depolarization. We conclude that in human B cells: (1) depolarization may be a "general response" to a variety of membrane stimuli, because F(ab')2 anti-mu and B-cell growth factor acting through different receptors both induce it, and (2) depolarization does not inevitably lead to cell volume increase.

Antigen-Antibody Reactions↗
PubMed1987

Warfarin-related purple toes syndrome and cholesterol microembolization.

The "purple toes syndrome" is a rare complication of oral anticoagulant therapy. Four patients who presented with "purple toes syndrome" several weeks after warfarin therapy was initiated are described. The diagnosis of cholesterol microembolization was made by biopsy in three cases. Malignant hypertension and renal failure developed in two patients who died within three to six months of onset of purple toes. Postmortem examination in one of these patients showed widespread cholesterol microembolization. Renal failure has not developed in the other two patients, who are doing well. These biopsy and autopsy results suggest that the warfarin-related "purple toes syndrome" is due to cholesterol microembolization.

Aged↗
PubMed1987

Abnormal expression of surface immunoglobulin isotypes on antigen-binding B lymphocytes from mice tolerant to trinitrophenyl determinants.

Temporary B-cell tolerance to the trinitrophenyl (TNP) hapten can be produced in BDF1 mice by intraperitoneal injection of trinitrobenzene sulfonic acid (TNBS). Antigen-binding cells (ABC) specific to TNP, measured as TNP donkey erythrocyte rosettes, are found in tolerant mice as well as in immune mice. We have studied the surface immunoglobulin isotype profile of these TNP-binding lymphocytes (TNP-ABC) in four groups of animals: nonimmune, immune, tolerant, and tolerant-challenged. Immune mice received intravenous TNP sheep erythrocytes (TNP-SRC), whereas tolerant-challenged mice received TNP-SRC and TNBS on Day 0. TNP-ABC from mice immunized with TNP-SRC exhibit increased expression of surface IgG and decreased expression of surface IgD, compared to the ABC from nonimmune mice. Tolerant mice have a higher proportion of ABC with surface IgG, and a lower proportion with surface IgD, than nonimmune mice. Tolerant-challenged mice have a lower proportion of ABC with surface IgG, and a higher proportion with surface IgD, than immune mice. Thus, B-cell tolerance in this model entails an attenuation of the surface immunoglobulin isotype switch (loss of IgD and gain of IgG) on ABC seen in the normal immune response. For most TNP-ABC, tolerogen exposure prevents the switch in surface isotypes normally induced by exposure to TNP antigen; i.e., the tolerance lesion precedes the surface isotype switch. However, a minority of the TNP-ABC appear to switch surface isotypes in response to the tolerogen itself.

Animals↗
PubMed1986

Mitogen-stimulated phospholipid synthesis in normal and immune-deficient human B cells.

Eight patients with common variable panhypogammaglobulinemia were shown in the in vitro Ig biosynthesis assay to have defective B cell responses to pokeweed mitogen (PWM). Phospholipid synthesis was assessed in the B cell plus monocyte fraction (MB) and irradiated T cells (T*) of patients and paired normal controls. Cell populations were studied separately and in the four possible combinations (1:1), with and without PWM, to reveal the effect of cell interactions. At 16 to 20 hr the mean stimulation index (SI) +/- standard error for MB cells alone was 1.01 +/- 0.02 for eight patients and 0.99 +/- 0.02 for the paired normals; the T* cell SI was 1.25 +/- 0.04 for patients and 1.28 +/- 0.05 for normals. Combinations of normal MB cells with normal T* cells showed significantly higher SI when compared with the combinations of normal MB cells with patient T* cells (p less than 0.005). However, the combination of patient MB cells with patient T* cells and the combination of patient MB cells with normal T* cells were not significantly different in SI (0.05 less than p less than 0.1). Isolation of patient and normal B cells, T* cells, and monocytes after the choline pulse showed that patient B cells gave a higher SI with normal T* help than with patient T* help. Of greatest interest is the finding that patient B cells that were defective in PWM-stimulated Ig production nevertheless showed a phospholipid synthesis response to PWM in the normal range, suggesting that the maturation defect in these B cells occurs later than the phospholipid synthesis acceleration step, or on a different pathway.

Adolescent↗
PubMed1986

Membrane defects of the tolerant B cell. IV. Failure to accelerate antigen receptor loss.

Mechanisms of immunologic tolerance affecting antibody responses include conditions extrinsic to the B cell such as dominant suppression by T cells (1), regulation by anti-idiotype (2), and tolerance in T helper cell populations (3). But tolerance can also result from changes in the antigen-reactive B cells such as their deletion (4), or that mysterious process by which they become "intrinsically tolerant", i.e., refractory to stimulation (5). One approach to learning more about the mechanism of intrinsic tolerance at the level of cell physiology is to determine which of the activation events that normally follow antigen contact occur or fail to occur in such cells. An established model of intrinsic B cell tolerance previously exploited in such studies in the trinitrophenyl (TNP)-self-induced tolerance model of Fidler and Golub (6). Having established that BDF1 mice injected with 2,4,6-trinitrobenzene sulfonic acid (TNBS) become tolerant to TNP, they showed by appropriate transfer experiments that the tolerance could be not induce antibody to TNP in such mice (8). cells (7). They also showed that lipopolysaccharide could not induce antibody to TNP in such mice (8). Together, these data indicated that in this example, tolerance is intrinsic to the B cells. B cells with receptors for TNP remain in these mice (9), providing an opportunity to study activation events in intrinsically tolerant B cells. This paper is part of an ongoing series of studies of activation events in TNP-antibody-binding cells (ABC)2 using this tolerance model (9-11). It shows that a TNP-antigen that normally induces rapid loss of antigen receptors on TNP-ABC cannot do so in mice rendered tolerant to TNP.

Animals↗
PubMed1986

The effects of nitrogen mustards on the proliferative and Ig synthetic response of human peripheral blood lymphocytes.

Maximal inhibition of pokeweed mitogen-stimulated Ig production and [3H]thymidine incorporation was shown to occur when unfractionated human peripheral blood mononuclear cells were cultured with concentrations of the nitrogen mustards melphalan, mechlorethamine or chlorambucil in the 20-100-microM range, whereas concentrations of microsome-activated cyclophosphamide (A-Cy) in the 2-mM range were required for equivalent inhibition. Around 400 microM A-Cy, IgM secretion was not inhibited, but secretion of IgA and IgG was. The [3H]thymidine incorporation of enriched populations of both large and small B and T cells all showed about 20-50-fold greater sensitivity to melphalan than to A-Cy, despite a difference of only 6-fold in alkylating activity between these drugs. Large (250 micron 3) B and T cells were only marginally more sensitive to melphalan and A-Cy than small (210 micron 3) T and B cells. Kinetic studies showed that IgG and IgA secreted by day 7 could be maximally inhibited by melphalan added as late as day 3, and IgM synthesis as late as day 2. In contrast, inhibition of Ig production by A-Cy steadily declined after the first day, especially IgM, which was no longer inhibitable by A-Cy on day 3. Inhibition of cumulative Ig production did not occur when A-Cy or melphalan was added on day 5 or later. Cell recombination experiments performed with drug pulsed and untreated monocytes plus B cells and irradiated T cells showed that inhibition of [3H]thymidine or Ig production was most striking when monocytes + B cells (rather than T cells) were exposed to melphalan in the first 16 h. When A-Cy was used in the first 16 h, inhibition of Ig production was partial and inconsistent, and inhibition of monocytes + B cell or T cell [3H]thymidine incorporation was not evident. We conclude that the nitrogen mustards melphalan and A-Cy can inhibit pokeweed mitogen-stimulated DNA synthesis by human T or B cells and Ig production in vitro, but that their mechanisms of action differ.

Adult↗
PubMed1985

Expansion and cell surface Ig isotype switching in the antigen-binding cell population of nu/nu mice and nu/+ littermates.

Swiss-Webster nu/nu splenocytes placed in modified Mishell-Dutton culture containing sheep red blood cells (SRC) generated increased numbers of antigen-binding cells (ABC) compared with antigen-free cultures. In contrast Balb/c nu/nu cultures did not expand their ABC population in response to SRC, suggesting that strain background may influence the effect of the nu/nu gene on T-dependent immune responses. Cell surface Ig isotype analysis indicated that the SRC-induced expanded ABC population exhibited a significant decrease in cell surface IgD and a significant increase in ABC bearing both IgM and IgG. The Swiss-Webster nu/+ littermate cell surface Ig isotype patterns were generally similar to the nu/nu ABC patterns, but with different kinetics.

Animals↗
PubMed1985

Early protein synthesis in activated human peripheral blood mononuclear cells.

Though B-cell division and Ig synthesis in response to pokeweed mitogen (PWM) require interaction with T-cells and monocytes, it is not clear which earlier events in B-cell activation share this requirement, and which are the result of direct interaction of mitogen with the B-cell. Having previously shown that the acceleration of lecithin synthesis in human B-cells at 16-20 hr requires both T-cells and monocytes, we now examine whether B-cells require similar interactions to increase their protein synthetic rate, another important activation event. At 21-24 hr of PWM stimulation, the stimulation index (SI) for incorporation of [35S]methionine into protein was 2.1 +/- 0.4 for unfractionated cells, 1.7 +/- 0.1 for B-cells, 2.5 +/- 0.1 for T-cells, and 3.4 +/- 0.5 for monocytes. Thus monocytes contributed substantially to early mitogen-induced protein synthesis by human peripheral blood mononuclear cells. When the monocyte/B-cell fraction (MB) and T-cell fraction (T) were mixed at various ratios in PWM-stimulated cultures, synergy was apparent at MB:T ratios of 1:1 and 1:2, indicating cell interactions augmented early mitogen-driven protein synthesis in at least one of these cell types. However, much or all of this synergy could be attributed to T-cells, whose protein synthetic response was augmented by B-cells and monocytes. In contrast, the early increase in B-cell protein synthesis appeared to be independent of cell interactions, since their SI of 1.7 was not influenced by varying the proportion of M- or T-cells over a 50-fold range. These contrasting results between two contemporary events fits the hypothesis that one (accelerated phospholipid synthesis) requires a first signal plus one or more cell interaction signals, whereas the other (accelerated protein synthesis) requires only the first signal.

Adult↗
PubMed1985

Antigen-induced acceleration of shedding and replacement of B cell antigen receptors requires mature T cells.

To determine which early and intermediate events in the response of antigen-binding B cells to a T-dependent antigen (sheep erythrocytes [SRC]) require T help, the antigen-induced changes in receptor turnover and surface IgD loss in BALB/c athymic nu/nu mice were compared with that of nu/+ littermates and +/+ BALB/c mice. Nonimmune SRC antigen-binding spleen B cells (ABC) from +/+, nu/+, and nu/nu BALB/c mice coexpressed IgM and IgD, and 85 to 95% retained receptors well when incubated for 2.5 hr in 100 micrograms/ml cycloheximide (which prevents receptor replacement). Also they were able to regain their ability to bind antigen by 18 hr after pronase treatment, but not by 2 hr. However, 5 days after in vivo immunization, 1) the proportion of ABC expressing surface IgD declined from around 90% to less than 50% in +/+ mice and nu/+ mice but not in nu/nu mice; 2) substantial recovery of antigen-binding occurred by 2 hr after pronase treatment in +/+ and nu/+ ABC but not in nu/nu ABC; and 3) when spleen cells were incubated in cycloheximide, uncompensated receptor shedding reduced +/+ and nu/+ ABC by around 80% but produced only about a 10% reduction in nu/nu ABC. Thus, although the ABC in nonimmune nu/nu mice appeared normal with respect to their surface Ig turnover and expression, they failed to undergo the normal antigen-induced loss of IgD or acceleration of surface Ig shedding and replacement, suggesting that these intermediate activation events require interaction with mature T cells. To determine whether this interaction had to occur during B cell development, during the development of the immune response, or during receptor shedding or replacement itself, cell transfer experiments were carried our wherein nu/+ T cells were transferred i.v. to nu/nu littermates 1 day before immunization with SRC. In the transfer recipients, pronase-treated day 5 ABC were then able to replace and shed their receptors at the accelerated rate, like ABC from +/+ and nu/+ mice. In contrast, the co-incubation of 5-day immune nu/+ T cells with nu/nu B cells did not alter the rate of shedding or replacement.

Animals↗
PubMed1984

Rapid separation of human monocytes and lymphocytes by Sephadex G-10.

Many of the interesting functions of human lymphocytes in vitro are known or suspected to be dependent on the presence of monocytes. A practical limiting factor in cell interaction experiments is the ability to separate and recover functional monocytes and lymphocytes rapidly from the same blood specimens. Here we describe a simple, rapid and inexpensive solution to this problem. Incubation at 37 degrees C on Sephadex G-10 columns consistently depleted monocytes to low levels (less than 0.1%) and gave maximum lymphocyte yields (greater than 60%) in a short time. Half the esterase-positive monocytes entering the column may be easily recovered from the beads by a 4 degrees C chilling/shaking technique; by using 0.5% lidocaine, an additional 15% can be recovered. Simultaneously, the G-10-nonadherent lymphocytes are being further separated into T and B cells by E-rosetting. These recovered G-10-adherent cells were above 90% esterase positive, and showed a cell size distribution on the Coulter channelyzer distinctly larger than nonadherent lymphocytes. To illustrate the use of this protocol we present a monocyte titration experiment which demonstrated a plateau of optimal pokeweed mitogen-stimulated Ig production at 5-10% monocytes.

Adult↗
PubMed1984

IgG aggregates of different sizes stimulate or suppress Ig secretion by human lymphocytes in vitro.

The regulatory effects of IgG aggregates on Ig production in vitro by human peripheral blood lymphocytes were shown to be highly dependent on the aggregate size and the degree of mitogenic stimulation. Covalently linked oligometers of IgG were prepared with dimethylsuberimidate cross-linking and chromatographic separation; larger aggregates were prepared by heating (63 degrees C) and preparative zonal ultracentrifugation. The storage and culture conditions used were shown to preserve the stability of aggregate sizes. Although both positive and negative regulatory effects were seen with cells isolated directly from blood, more predictable dose-related effects were seen if cells were vigorously washed, possibly due to the removal of IgG or natural immune complexes bound by the cells in vivo. Some preparations of small IgG oligomers produced marked stimulation of Ig production, especially in cells cultured without mitogen or with suboptimal pokeweed mitogen doses. Aggregates containing six or more IgGs suppressed Ig production, especially when cells were stimulated by mitogen at optimal concentrations.

Antigen-Antibody Complex↗
PubMed1984

Phospholipid transmethylation in human mononuclear cells is not influenced by mitogens.

The importance of accelerated methyl transfer from methionine to membrane phospholipid (PL) as an early event in lymphocyte activation is vigorously debated. We have examined pokeweed mitogen (PWM)- and phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes for this early activation event. Without mitogen, methyl groups rapidly entered into the PL of unfractionated human lymphocytes by 10 min, and then adopted a rate which stayed constant for at least 2 hr. Whether or not mitogen was added after a 60-min preincubation, with [3H]methyl methionine, after a further 20 min, Sephadex G-10 adherent monocytes were incorporating methyl groups into PL 4 times faster than B-cells and 9 times faster than T-cells. At 10(7) cells/ml, neither PWM nor PHA changed total PL labeling kinetics, or produced a significant change of methyl groups from phosphatidyl-N-mono- and dimethylethanolamine to phosphatidylcholine or lysophosphatidylcholine in B-cells, T-cells or monocytes, whether these populations were cultured separately or together. Even for the slowly incorporating human T-cells, the background rate of methyl transfer per cell was much greater than that previously reported for mouse or pig mononuclear cells. Accelerated methylation of phospholipid appears not to be a favorable early event by which to study human lymphocyte activation, and we agree with Moore et al. [J. biol. Chem. 257, 8183-8189 (1982)] that it may not be a universal activation event.

Cell Communication↗
PubMed1983

Antibody-secreting cell precursor frequencies among the sheep-erythrocyte-binding cells after immunization.

Controversy concerning the immunologic role of antigen-binding cells (ABC) has prompted us to attempt to quantitate the proportion of stimulable ABC, in immunized animals, which are precursors for cells producing antibody specific for the antigen bound. Using a lipopolysaccharide (LPS)-driven limiting dilution analysis system, the precursor frequency (PF) of cells secreting IgM and IgG and sheep erythrocyte (SRBC)-specific IgM and IgG was established for highly purified SRBC antigen-binding cell (SRBC-ABC) and unfractionated populations taken from CBA/J mouse spleens on days 5, 12 and 180 of the in vivo primary immune response to SRBC. At all these times, almost all SRBC-ABC spontaneously secreting immunoglobulin (Ig) secreted SRBC-specific Ig, and almost all precursors of Ig-secreting cells in the ABC populations were precursors of cells secreting specific anti-SRBC antibody. In SRBC-ABC populations, the PF for total and SRBC-specific Ig secretion was seen to decrease on days 5 and 12 after immunization and to increase to 3.5 to 7 times nonimmune levels 180 days after immunization. The absolute number of precursors, within the SRBC-ABC population, for the secretion of SRBC-specific Ig decreased on day 12 after immunization. In the unfractionated population, the PF for SRBC-specific Ig secretion temporarily increased after immunization, reaching peak levels 5 days (IgM) and 12 days (IgG) after immunization. These two changes may be related, representing the progress of stimulated cells out of the ABC pool as they lose receptors en route to full maturation. The small clone sizes on days 5 and 12 indicate that ABC divide less in response to LPS when already engaged in a response to antigen. In contrast, the PF for total IgM and IgG secretion in the unfractionated population was not greatly affected by immunization.

Animals↗
PubMed1983

Phospholipid synthesis by activated human B lymphocytes.

Pokeweed mitogen- (PWM) stimulated DNA and Ig synthesis in human B cells is dependent on the presence of T cells and adherent cells, but the influence of these regulatory cells on earlier activation events is unknown. We have studied the T cell and monocyte influence on the incorporation of [methyl-14C]choline chloride into B cell phospholipids (PL) after varying periods of in vitro culture with or without pokeweed mitogen (PWM). By separating B and T cells after choline pulsing, a peak in PWM-induced PL synthesis of B cells at days 1 to 2 was revealed, whereas the T cell response was later (days 2 to 3). In the first 4 hr of culture, the purified B cell plus monocyte fraction incorporated choline four to six times faster than the T cell fraction, but PWM did not increase choline incorporation, whether these fractions were cultured separately or together. When cultures were pulsed with choline between 16 and 20 hr with or without PWM, monocytes incorporated choline six to nine times faster than T cells, and B cells were intermediate. Also at 16 to 20 hr of culture, a significant PWM-induced increase in choline incorporation by B cells was evident and was dependent on the presence of T cells and monocytes. The monocytes showed no increased choline incorporation due to PWM. Thus, the influence of regulatory cells on the PWM response in B cells is evident within the first 24 hr.

Adult↗
PubMed1982

Panhypogammaglobulinemia in systemic lupus erythematosus: in vitro demonstration of multiple cellular defects.

Classically, systemic lupus erythematosus (SLE) is a disease of antibody overproduction, whereas the hallmark of acquired immune deficiency is antibody underproduction. Two patients are presented in whom panhypogammaglobulinemia developed during the course of SLE. In both patients, the levels of the major immunoglobulin (Ig) classes did not fall simultaneously. Anti-DNA antibodies were present, and exacerbations of SLE nephritis occurred in both cases 6 to 8 yr after Ig levels became subnormal. One patient still requires immunosuppressive therapy for renal disease; both patients are experiencing recurrent sinopulmonary bacterial infections. In the pokeweed mitogen--stimulated Ig biosynthesis assay, both patients showed abnormal Ig production due to defective function of three cell types: hyporesponsive B cells, excessive T suppression, and subnormal T help. The latter defect is rare in common variable hypogammaglobulinemia. One patient also showed extreme suppression of Ig production by phagocytic mononuclear cells. Thus, despite the similarity in the histories, the cellular function of these two patients was not identical in vitro.

Adult↗