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S J Ewald

Publications and source records attributed to S J Ewald.

At least 37 records · Page 2Linked to original sources
PubMed1993

Ethanol increases apoptotic cell death of thymocytes in vitro.

Exposure of animals to ethanol causes thymic atrophy in adults and fetuses. Whether direct effects of ethanol contribute to thymic atrophy or whether indirect effects are entirely responsible is at present unknown. In the normal animal, large numbers of thymocytes undergo a physiological form of cell death referred to as "apoptosis." To determine if ethanol affects the process of apoptosis, studies were undertaken in which mouse thymocytes were cultured overnight in the presence or absence of ethanol. Apoptotic cell death was analyzed by flow cytometric quantitation of apoptotic nuclei, by fluorometric measurement of DNA fragments, and by gel electrophoretic analysis of DNA fragments. Ethanol in concentrations of 0.2% to 0.8% produced significantly higher levels of apoptosis than were seen in control cultures. The DNA fragmentation was characterized as apoptotic on the basis of inhibition by aurintricarboxylic acid (an inhibitor of nucleases) and by the presence of characteristic oligonucleosomal-sized fragments of DNA. The effect of ethanol on apoptosis was additive to that induced by immobilized anti-CD3 monoclonal antibody. CD4+CD8+ cells underwent apoptosis as indicated by reduction in CD4 and CD8 surface antigen expression. An inhibitor of protein kinases (H-7) reduced the DNA degradation induced by ethanol and by anti-CD3. These results suggest that direct effects of ethanol contribute to thymic atrophy in alcohol-consuming animals.

Animals↗
PubMed1992

Characterization and differential expression of protein kinase C isoforms in PC12 cells. Differentiation parallels an increase in PKC beta II.

Nerve growth factor (NGF) treatment of PC12 cells induced a 2.8-fold increase in protein kinase C activity concomitant with differentiation and acquisition of neuritis. PKC protein isoforms were separated by sequential chromatography on DEAE-Sephacel/hydroxylapatite. A broad peak of PKC activity eluted which corresponded to the alpha PKC isoform. In control cells, message for all six PKC isoforms was detected and expressed as epsilon greater than zeta = gamma greater than delta greater than beta greater than alpha. Western blot of whole cell lysates revealed a large increase in the beta II, while slight changes were observed for the other five PKC isoforms during treatment (1-14 days) with NGF (50 ng/ml). In parallel, coordinate changes in the expression of the individual transcripts for the six isoforms occurred during NGF treatment. Induction and accumulation of PKC beta II may play a role in maintenance of neuronal morphology.

Animals↗
PubMed1991

Alcohols synergize with NGF to induce early differentiation of PC12 cells.

The role of alcohols in affecting neuromorphogenesis was investigated in a single cell type, pheochromocytoma (PC12). The effect of ethanol at physiological concentrations in this system leads to enhanced morphological and functional differentiation in combination with nerve growth factor (NGF). PC12 cells treated with a suboptimal concentration of NGF (30 ng/ml) and an alcohol (87 mM) underwent rapid morphological differentiation which was dependent upon the side chain length of the alcohol MeOH less than EtOH less than PrOH less than BuOH. Pyrazole at either 5 or 10 mM had no effect on alcohol induced neurite extension. Assessment of the degree of differentiation promoted by the various alcohols was quantified by an increase in neurite extension, a decrease in the incorporation of [3H]thymidine, an increase in acetylcholine esterase (AChE) activity and immunostaining with neuron specific enolase. Thus, alcohols may function in a specific manner by interacting with transmembrane signalling pathways which promote gene expression and neuronal differentiation.

Acetylcholinesterase↗
PubMed1989

Concanavalin A induces a cytoskeletal association of T200 molecules in T lymphocytes.

A recent report indicated that T200 molecules interact with elements of the cytoskeleton in BW5147 T lymphoma cells. We have confirmed the cytoskeletal association of T200 by examining nonionic detergent-soluble and detergent-insoluble fractions of murine T cell tumor cell lines, cloned cytotoxic T lymphocyte lines, and thymocytes. Concanavalin A (Con A)-treated and untreated cells were extracted with 0.5% Triton X-100 and the remaining insoluble material was extracted under conditions allowing actin depolymerization. In the absence of Con A treatment, little T200 could be recovered from the depolymerized insoluble fraction. However, in T cells treated with capping concentrations of Con A, a considerable amount of T200 was rendered insoluble in nonionic detergent, and T200 could be recovered from the insoluble fraction by a buffer which dissociates actin polymers. A lesser, but still significant, amount of T200 associated with the detergent-insoluble fraction of thymocytes treated with concentrations of Con A and succinyl Con A, which are mitogenic for T cells. We also found that in T cells treated with mitogenic concentrations of succinyl Con A, more T200 associated with cytoskeleton than did H-2 or LFA-1 molecules. Because T200 is such a predominant molecule on the surface of T cells, such translocations of the molecule may have a major impact on the physiology of the cell, especially if T200 functions as a protein tyrosine phosphatase as recent evidence by others suggests.

Animals↗
PubMed1989

T lymphocyte populations in fetal alcohol syndrome.

There are reports that fetal alcohol syndrome (FAS) is associated with immune deficiency or DiGeorge syndrome. To investigate the effect of prenatal alcohol exposure on the immune system, we used a mouse model of FAS in which C57BL/6J female mice were fed a complete liquid diet containing 25% ethanol-derived calories (EDC) from gestational day (g.d.) 1 to 18. Thymus cell numbers were markedly reduced in 18-day fetuses exposed to ethanol. Thymocytes from fetuses from the 25% EDC diet group and from pair-fed and ad-libitum control diet groups were compared by flow cytometry for expression of T cell differentiation antigens. The proportions of L3T4- and Lyt-2 positive thymus cells were significantly reduced in alcohol-exposed fetuses compared to controls; however, the number of Thy-1-positive cells did not differ among any of the groups. Six-day old neonates exposed prenatally to ethanol from g.d. 1 to 13 had thymus and spleen T cell populations similar to those of controls in almost all cases, indicating a "catch-up" of T cell numbers in most animals. Spleen T cell function, assessed by response to Concanavalin A (Con A), or Con A plus T cell growth factors, was somewhat depressed in ethanol-exposed 6-day pups.

Animals↗
PubMed1988

Flow cytometric and histological analysis of mouse thymus in fetal alcohol syndrome.

Reduced thymus size and fetal weight were seen in 18-day old fetuses of C57BL/6 female mice fed a complete liquid diet containing 25% ethanol-derived calories (EDC) from gestational day 0 to 18. Thymocytes from fetuses from the 25% EDC diet group and from pair-fed and ad-lib control diet groups were compared by flow cytometry for expression of thymocyte differentiation antigens. The proportions of L3T4-positive and Lyt-2-positive thymus cells were significantly reduced in alcohol-exposed fetuses compared to controls; however, the number of Thy-l-positive cells did not differ among any of the groups. Histologically, the thymus from 25% EDC fetal mice failed to show the delineation between cortex and medulla that was seen in the thymuses of control fetuses. These results indicate that thymus immaturity is one of the accompanying features of fetal alcohol syndrome.

Animals↗
PubMed1988

Effect of Ly 5 allotype on in vitro immune responses.

We have tested in vitro immune responses of several kinds to determine if Ly 5 allotype influences reactivity of murine splenocytes in processes thought to involve the T200 glycoprotein. Matings were established among C57BL/6J (B6) (Ly 5.1) and C57BL/6-Ly 5.2 (B6-Ly 5.2) congenic mice (both H-2b) to obtain sibling mice segregating for alloalleles of the Ly 5 system. F2 progeny of the three Ly 5 genotypes were tested for antibody-dependent cell-mediated cytotoxicity (ADCC), proliferation in allogeneic mixed lymphocyte culture (MLC), mitogen responsiveness, natural killer (NK) cell activity, and cytotoxic T-lymphocyte (CTL)-mediated cytotoxic activity. We observed that in Ly 5 segregant mice, higher CTL activity was associated with the Ly 5.2 type. No allotype effect was observed in MLC, mitogen responses, and NK cell-mediated cytolysis. In the parental and F1 animals, mice carrying the Ly 5.2 allele had significantly higher ADCC levels, though this effect was not seen in the segregants. Our results indicate that Ly 5 or some closely linked gene or genes influence CTL activity of murine splenocytes in vitro.

Animals↗
PubMed1987

Inhibition of in vitro generation of cytotoxicity against tumor cells by monoclonal antibody to Thy-1.

The Thy-1 molecule on murine T lymphocytes has been suggested to play a role in cellular activation events leading to a variety of immunologic functions. We present evidence that this molecule may be involved in signals leading to the in vitro generation of cytotoxic T cells against several tumor cell lines used as stimulators in mixed tumor-lymphocyte culture. The presence of monoclonal antibody against a polymorphic determinant on the Thy-1 molecule markedly reduced the generation of cytotoxicity after three days of culture of murine splenocytes with stimulator tumor cells bearing low levels of Ia antigen. In contrast, no effect was seen when the stimulators were either allogeneic splenocytes, or a tumor cell line expressing large amounts of Ia. These results suggest that the Thy-1 molecule is critically involved in events leading to the generation of cytotoxic effectors under some, but not all conditions.

Animals↗
PubMed1987

Effects of Ly-5 antibodies on antibody-dependent cell-mediated cytotoxicity (ADCC).

Antibodies to a number of cell surface antigens expressed on mouse spleen cells were tested for their ability to inhibit antibody-dependent cell-mediated cytotoxicity (ADCC) of antibody-coated sheep erythrocytes (Ab-SE) in the absence of complement. Of the antibodies tested, only the antisera to Ly-5 and H-2 antigens significantly inhibited ADCC by spleen cells reactive with those antisera. Inhibition by Ly-5 antisera was shown to be allotype-specific by inhibition experiments using C57BL/6-Ly-5.1 (B6-Ly-5.1) and congenic C57BL/6-Ly-5.2 (B6-Ly-5.2) mice. Inhibition by Ly-5.1 antiserum appeared not to be due to competition for the Fc receptor (FcR), since in mixing experiments 'third-party' thymus cells treated with Ly-5 antiserum did not inhibit the cytotoxic activity of untreated cells. In comparing inhibition induced by antisera to H-2 and Ly-5 antigens, Ly-5.1 antiserum was more inhibitory at nearly every dilution tested. In addition, F(ab')2 fragments of Protein A-purified Ly-5.1 antibody were inhibitory to BALB/c spleen effector cells in ADCC of Ab-SE, whereas F(ab')2 fragments of H-2 antibodies had no effect. Because ADCC of tumour cells may share a common lytic mechanism with ADCC of erythrocytes, several antisera to cell surface antigens found on spleen cells were tested for inhibition of ADCC to antibody-coated P815 tumour cells (Ab-P815). As seen in ADCC to Ab-SE, anti-Ly-5.1 was a more potent inhibitor than antibody against either H-2k or H-2d antigens. The results are discussed in the light of the ability of Ly-5 antisera to inhibit various types of cell-mediated killing.

Animals↗
PubMed1987

Effect of prenatal exposure to ethanol on development of the thymus.

The effect of maternal alcohol consumption on development of the fetal thymus was studied in C57BL/6 mice. Female mice were fed a complete liquid diet containing 25% ethanol-derived calories (or a control diet) both before and during pregnancy. Significantly reduced fetal weight (a characteristic feature of fetal alcohol syndrome) was noted in 18- and 19-day-old fetuses of alcohol-fed mice. The thymuses of fetal mice exposed prenatally to ethanol were reduced both in cell number and proliferative response to Concanavalin A plus a source of interleukin 2.

Animals↗
PubMed1985

Analysis of structural differences between Ly-5 molecules of T- and B-cells.

A group of closely related high mol. wt (Mr) membrane glycoproteins is expressed with varying Mr on different subpopulations of lymphocytes, but the different Mr forms share the Ly-5 and T200 antigenic determinants. The Ly-5 molecule expressed by thymocytes has an Mr of 175,000 (175ly5). The antigenically related molecule on B-cells has an Mr of 210,000 (210ly5). It is not known whether the variations in size are due to differences in the polypeptide chain, post-translational modifications such as glycosylation, or both. In this report we examine the glycosylation of 175ly5 and 210ly5 to determine whether differences in carbohydrate moieties may account for the different Mr of these two Ly-5 species. Pronase digestion and alkaline borohydride treatment of these molecules labeled in the terminal galactose residues revealed that 210ly5 molecules have a more complex oligosaccharide pattern than 175ly5 molecules. While Ly-5 oligosaccharides from a T-cell tumor line were very similar to those of normal thymocytes, the pattern of Ly-5 carbohydrates from a B-cell tumor were somewhat different than those from normal B-cells. This report also presents evidence for O-linked sugars on Ly-5 molecules.

Animals↗
PubMed1985

Co-immunoprecipitation of the Ly-5 molecule and an endogenous protease: a proteolytic system requiring a reducing agent and Ca2+1.

Sodium [3H]borohydride- and [35S]methionine-labeled Ly-5 molecules from mouse thymocytes and T lymphoma cells were isolated with specific antibody and Staphylococcus aureus Cowan I (SaCI) strain; after extensive washing of the complexes, elution with Laemmli's reducing buffer (0.05 M Tris [pH 6.8 or 6.0], 4% sodium dodecyl sulfate [SDS], and 2% 2-mercaptoethanol [2-ME]) resulted in partial breakdown of the isolated Ly-5 molecules from a Mr = 175,000 to 150,000. Other proteins present during the elution step showed no evidence of proteolysis. 2-ME and SDS were required for proteolysis; although addition of exogenous Ca2+ during elution was not necessary, both EDTA and EGTA inhibited breakdown of the molecule that could be overcome by excess Ca2+. Of a variety of protease inhibitors and thiol-reactive agents tested, only TAME and oxidized glutathione blocked proteolysis almost completely. SaCI, serum, and contaminating antibodies were ruled out as the source of the proteolytic activity. More stringent preclearing and washing conditions did not eliminate endogenous proteolysis of the Ly-5 molecule. The endogenous proteolytic fragment had a Mr distinct from the tryptic fragment of the Ly-5 molecule. We conclude that the Ly-5 molecule may be autolytic or tightly associated with a distinct cellular protease.

Animals↗
PubMed1984

Inhibition of T cell responses to alloantigens and polyclonal mitogens by Ly-5 antisera.

The effects of Ly-5 alloantisera on the generation of cytotoxic T cells (CTL), on the effector phase of CTL killing, and on polyclonal mitogenesis were studied. Ly-5 antisera added at the beginning of mixed lymphocyte culture (MLC) suppressed the production of CTL in an allele-specific manner. Neither Ly-5.1 nor Ly-5.2 antisera inhibited the generation of cytotoxic effectors by Ly-5.1/Ly-5.2 heterozygous spleen cells; however, a combination of Ly-5.1 and Ly-5.2 antisera markedly suppressed the appearance of Ly-5 heterozygous CTL. Similarly, Ly-5 antisera inhibited the effector phase of CTL killing in an allele-specific manner. In addition, Ly-5 alloantisera specifically blocked concanavalin A and oxidative mitogenesis of splenocytes carrying the appropriate Ly-5 alloantigen. The results are discussed in light of a possible functional role of Ly-5 molecules in immune processes.

Animals↗
PubMed1983

Modulation of in vitro immune responses by monoclonal antibody to T200 antigen.

The effects of monoclonal antibody to the T200 antigen on murine mixed-lymphocyte cultures (MLC) and on the generation of alloreactive cytotoxic T lymphocytes (CTL) are investigated. Addition of monoclonal anti-T200 without complement to MLC results in a late suppression of the proliferative response preceded in some cases by an early enhancement. These modulations require the presence of allogeneic stimulator cells; no effects are seen when antibody is added to responders alone. A similar effect is seen on the generation of CTL. Compared to controls without antibody, cultures carried out in the presence of anti-T200 show reduced levels of cytotoxicity measured against allogeneic targets by Day 5. The kinetics of the suppressive effects differ from those seen with anti-Lyt-2, and no suppressive effects are seen with monoclonal antibodies to other cell surface molecules.

Animals↗
PubMed1981

Tryptic peptide map analyses of mouse transplantation antigens.

Tryptic peptide map analyses of five K- and three D-gene products of various H-2 haplotypes are presented. These data support earlier sequence studies and demonstrate that the variations in allelic gene products of the K or D loci are scattered throughout much of the polypeptide chains. Furthermore, the K allelic gene products are no more closely related to one another than they are to the D allelic gene products. This apparent lack of K-ness and D-ness places interesting constraints on the genetic organization and evolutionary history of the genes encoding the transplantation antigens.

Amino Acids↗
PubMed1980

Characterization of multiple immunoglobulin mu-chains synthesized by two clones of a B cell lymphoma.

We have identified 3 species of mu-chain synthesized by mouse WEHI 279 lymphoma cells that differ in cellular location, size and charge--mui (internal), mum (membrane), and mus (secreted). Lactoperoxidase and galactose oxidase labeling experiments localize the mum chain to the plasma membrane and the mui chain to the cytoplasm. Pulse-chase experiments demonstrate that the mui pool contains precursors to both mum and mus chains. Comparative peptide mapping studies and cell labeling in the presence of tunicamycin suggest that the mum chain is 2000 daltons larger than the mus chains and that the difference is due to polypeptide alterations rather than carbohydrate differences. The WEHI 279 lymphoma has differentiated while in culture to a more advanced stage in the pathway of B cell differentiation.

Animals↗