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Biomedical subjects

D A Lawrence

Publications and source records attributed to D A Lawrence.

At least 163 records · Page 9Linked to original sources
PubMed1986

Differential inhibition of human T-lymphocyte activation by maleimide probes.

Cellular thiols are known to be involved in lymphocyte activation, differentiation, and growth. In theory, alkylation of selective cellular thiols could be used to regulate specific processes in the activation sequence by inactivating particular enzymes or structural proteins, although to date specific alkylating probes have not been reported. N-Ethylmaleimide (NEM) is a lipophilic sulfhydryl-alkylating agent that is known to block the in vitro proliferative response of T lymphocytes. NEM (10 microM) was found to be fully inhibitory in PHA, Con A, and MLC assays only when added prior to or simultaneously with the mitogens or allogeneic cells; the addition of NEM only 15 sec after stimulating the cells with PHA resulted in a loss of greater than 50% of the inhibitory activity. The addition of 50 microM 2-ME 10 min after treating the cells with NEM failed to block the inhibitory effect. NEM (10-20 microM) had no adverse effect on lymphocyte viability, but completely blocked lymphocyte agglutination in response to mitogens or allogeneic cells. The lymphocytes overcame the inhibitory effects of NEM after 48 hr in both the PHA and MLC experiments. Resumption of the proliferative response was associated with the onset of agglutination in the PHA assay. In experiments using various analogs of NEM, we noted that the presence of a nonpolar N-linked side group was necessary for inhibitory activity. Pretreatment of PBMC with NEM decreased the total cellular thiols by 50% and blocked proliferation by 99%, whereas N-hydroxymaleimide decreased the total cellular thiols by 38% but had no effect on the proliferative response. The additional 12% of the cellular thiols that react with NEM, but not NHM, account for the inhibitory effect of NEM on lymphocyte proliferation. These findings suggest that selective cellular thiols are critical for T-cell activation.

Cytotoxicity, Immunologic↗
PubMed1986

Analysis of lead effects on in vivo antibody-mediated immunity in several mouse strains.

The effect of administration of lead acetate (10 mM in the drinking water) for 8 weeks on the in vivo sheep red blood cell (SRBC) specific plaque-forming cell (PFC) responses of inbred A, BALB/c, C57Bl/6, DBA/1, SJL, and NZW/NZB F1 mice and outbred CFW mice was examined to determine if lead was immunomodulatory in a genetically related manner. Lead did not suppress the SRBC-specific PFC/10(6) splenocytes or PFC/spleen response in any mouse strain when compared to the responses of strain-matched control mice. In addition, 10 mM lead-treated BALB/c mice manifested augmented PFC/10(6) splenocytes (17%; p less than .05) but unchanged PFC/spleen responses. Correspondingly, serum concentrations of SRBC-specific antibody (measured by radioimmunoassay) and serum immunoglobulin G, M, or A isotypes were also unchanged by lead acetate treatment in all tested mouse strains. There were no observable lead-related histopathological changes or deposition of immune complexes or antibasement membrane antibody in the kidneys of treated mice. Further, splenocytes from lead-treated, SRBC-immunized mice cultured with T-independent antigens (TNP-LPS, TNP-Ficoll) or with a T-dependent antigen (SRBC) exhibited direct and indirect specific PFC responses that were unchanged from those of control mice. The H-2K/D haplotypes of the outbred CFW mice were determined by microcytotoxicity to include r, q, u, and s. These results suggest that lead acetate (10 mM) administered po for 8 weeks does not suppress the primary direct humoral immune response to SRBC in inbred and outbred mice of several H-2 haplotypes (k/d; d; b; q; d,z; s; r; and u).

Administration, Oral↗
PubMed1986

T-cell chronic lymphocytic leukaemias and T-cell lymphoma-leukaemia: heterogeneity and anomalous cell markers.

We studied cell surface markers in 8 men with T-cell chronic lymphocytic leukaemia, using fluorescein-conjugated monoclonal antibodies and a cell analyser/sorter. 1 patient's cells had a T-suppressor/cytolytic phenotype (T4-, T8+) and in 3 patients the phenotype was a T-helper cell (T4+, T8-) as described in retrovirus-associated adult T-cell lymphoma-leukaemia. In 4 patients, the cell phenotype was anomalous, with both helper and suppressor antigenic markers (T4+, T8+). Terminal deoxynucleotidyl transferase was positive in 1 of the 4 cases with dual markers. Clinical features and cytological characteristics, including cell size and the density of T8 antigen on the cell surface, showed no obvious correlation with the immunophenotypes, but study of additional cases is needed to evaluate this further.

Aged↗
PubMed1985

Conversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions.

A latent beta-TGF activity is spontaneously released into serum-free culture medium by chicken embryo fibroblasts. Anchorage-independent growth activity measured on NRK-49F indicator cells, of this latent beta-TGF can be revealed by four different treatments: acidification, alkalinisation, exposure to urea, and heating to 100 degrees C for 3 minutes. This lact activating treatment indicates that latent beta-TGF activation in vitro is non-enzymatic. Active beta-TGF exists in a low molecular weight form 16 Kd (apparent) in 1M acetic acid, which elutes on reverse phase (FPLC) between 33-35% acetonitrile. Under neutral conditions only a high molecular weight form excluded on Biogel P60 is observed. This form is poorly active on NRK-49F for anchorage independent growth but can be fully activated by prior acidification. Rechromatography of the latent beta-TGF-containing fractions under acidic conditions converts the high molecular weight form to an apparent 16 Kd active form. We suggest that the high molecular weight form may correspond to a complex of a beta-TGF associated with a carrier or binding protein.

Animals↗
PubMed1985

Further study of beta-TGFs released by virally transformed and non-transformed cells.

Chicken embryo fibroblasts sensitized by ts RSV respond to TGFs present in the media of non-transformed FR3T3 and NRK-4 rat cells and of the same cells transformed by KiMSV or RSV. They also respond to TGFs present in the media of BHK hamster cells transformed by MoMSV, PyV or RSV. Two other indicator rat cell lines, untransformed NRK-4 and FR3T3, sensitized by ts KiMSV, respond to the same TGF-containing media, and this response is increased by exogenous EGF. Normal FR3T3 cells failed to respond to any of the media. The most sensitive target cells were the ts KiMSV-FR3T3 cells at the restrictive temperature (39.5 degrees C). All the media tested on NRK-49F target cells required EGF for their TGF activity which was essentially dependent on prior activation by acidification. These data show that the above media from non-transformed or transformed cells contain beta-TGFs, with no detectable accompanying alpha-TGF activity. The release of and the response to these TGFs are not interdependent. A function of ts mutant src and k-ras viral oncogenes, still expressed at the restrictive temperature, can sensitize non-responsive cells, without there being any specificity towards the TGF producer cells.

Animals↗
PubMed1985

2-Mercaptoethanol-dependent lipopolysaccharide-responsive B cells in nylon-wool-fractionated spleen cell preparations.

The ability of lipopolysaccharide (LPS) to influence lymphocyte mitogen responsiveness to 2-mercaptoethanol (2-ME) was investigated. 2-ME was mitogenic for splenocytes only when fetal bovine serum lots containing elevated endotoxin levels were used to supplement (5%) the culture medium. 2-ME did not stimulate mitosis of nylon-wool-fractionated splenocytes (NWSC); however, 2-ME induced significant proliferation in the NWSC cell preparations when endotoxin-deficient culture medium was supplemented with doses of LPS which alone did not stimulate significant proliferation. The sIg+ and Thy-1-/Lyt-1- fractions of NWSC, which represented less than 5% of the cells in the 'T-cell preparations', were almost completely dependent on 2-ME for proliferation in response to LPS stimulation. These data indicate that nylon-wool-fractionated preparations contain splenic B cells that incorporate significant amounts of [3H]thymidine when stimulated with LPS and 2-ME.

Animals↗
PubMed1985

Transforming growth factors--an overview.

Transforming Growth Factors (TGFs) are a sub-group of a larger family of protein hormones. Two major types of TGF are currently known, alpha-TGF and beta-TGF. Biologically their most important property is to act synergistically to induce anchorage-independent growth of target cells otherwise incapable of such growth. Since this growth parameter is well correlated with tumorigenicity in vivo, these factors may play a role in cancer development. Biochemically both alpha-TGF and beta-TGF have been well characterised in some types of cells and tissues. Their role in normal and neoplastic growth is actively studied.

Animals↗
PubMed1984

Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells.

Cultured bovine aortic endothelial cells are associated with an unusually stable fibrinolytic inhibitor (Loskutoff, D.J., van Mourik, J.A., Erickson, L.A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2956-2960). This inhibitor was purified to apparent homogeneity from medium conditioned by these cells by a combination of concanavalin A affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a single-chain glycoprotein of apparent Mr 50,000 +/- 2,500 and isoelectric point of 4.5-5.0, and inhibits the ability of both urokinase and tissue-type plasminogen activator to cleave and active plasminogen. This inhibition of plasminogen activator activity is associated with the formation of an enzyme-inhibitor complex which can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified inhibitor retains full activity after incubation in the presence of 0.1% sodium dodecyl sulfate, or at pH 2.7, two treatments which rapidly destroy the activity of protease nexin, another cellular inhibitor of fibrinolysis. The inhibitor purified from cloned endothelial cells cultured in the presence of L-[3,4,5-3H]leucine represented 2.5-12% of the total radiolabeled protein released by the cells in a 24-h period. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a protein which inhibits plasminogen activators and is distinct from protease nexin. It is a major endothelial cell product, and, as such, probably plays an important role in regulating the fibrinolytic system of these cells.

Animals↗
PubMed1984

Normal embryo fibroblasts release transforming growth factors in a latent form.

Normal chicken, mouse, and human embryo fibroblasts release into their culture media transforming growth factors (TGFs) in a latent form. Their soft agar colony-forming activity on two widely used target cells, rat NRK-49F and mouse AKR-2B, is essentially revealed only after prior acidification of cell-conditioned media. These TGFs are EGF-dependent when assayed on NRK-49F cells and EGF-independent on AKR-2B cells. The TGF activity from the chicken source is released in three (apparent) molecular weight forms of 500 kd, 125 kd, and 20 kd.

Animals↗
PubMed1984

Reverse fibrin autography: a method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate--polyacrylamide gel electrophoresis.

A new technique, reverse fibrin autography, was developed to detect protease inhibitors previously fractionated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Exogenous proteases were incorporated into fibrin-agar indicator films, eventually causing the fibrin to lyse. When an acrylamide gel containing inhibitors was placed on top of such an indicator, the positions of the inhibitors were revealed by the formation of opaque, lysis-resistant zones in the otherwise cleared fibrin film. The technique was versatile in that a variety of inhibitors were revealed, and semiquantitative since the size of the lysis-resistant zone in the indicator increased in proportion to the amount of inhibitor subjected to electrophoresis. This approach could be used not only to detect inhibitors having different protease specificities, but also to distinguish between the inhibitor activities of antibodies directed against urokinase or tissue-type plasminogen activator. Thus, reverse fibrin autography offers a convenient new approach to rapidly screen and partially characterize inhibitors present in complex biological samples.

Animals↗
PubMed1984

Latent beta-transforming growth factor in nontransformed and Kirsten sarcoma virus-transformed normal rat kidney cells, clone 49F.

Normal rat kidney cells of the clone 49F and their Ki-MSV-transformed counterparts spontaneously release the same transforming growth factor (TGF) activity in an inactive form. By acidification followed by neutralization prior to assay, this TGF activity is unmasked and promotes anchorage-independent growth of the NRK-49F indicator cells in the presence of epidermal growth factor. The TGF activity released by both cell types has an apparent molecular weight of 9,000 under acidic conditions, does not compete for binding to epidermal growth factor receptors, is heat resistant but dithiothreitol and trypsin sensitive, and therefore is of the beta-TGF class.

Animals↗
PubMed1984

The allogeneic effect: the mechanism of allosuppression by Lyt-1, Ia- T cells.

The kinetics of allohelp mediated by diffusable factors revealed that help by nonirradiated T cells (TOR) peaked at 48 to 72 hr, followed by a sharp decline if the T cells remained in the cultures. The temporal decrease in help after 72 hr was not mediated by suppressor lymphokines because mixtures of early (24 to 48 hr) and late (120-hr) allogeneic supernatants enhanced help synergistically. Lyt-1, Ia- T cells mediated the temporal decline in help and suppressed allogeneic B cell activation in co-cultures, and this "down-regulatory" activity (allosuppression) was radiosensitive. Help by irradiated T cells (T1000R) increased gradually until it plateaued between 96 and 120 hr. The helper activities of the allogeneic supernatants were directly proportional to their T cell growth factor (TCGF) activities. In addition, their kinetics were identical, and the removal of TCGF from 48-hr allogeneic supernatants by adsorption with TCGF-dependent HT-2 cells depleted both helper and TCGF activities. Help was restored to depleted 48-hr and 120-hr allogeneic supernatants by preparations of TCGF obtained from concanavalin A (Con A)-stimulated FS6-14.13 hybridoma cells that were adsorbed with lipopolysaccharide (LPS)-activated B cells or normal spleen cells (NS), but not with HT-2 cells. These results indicate that allohelp is dependent on TCGF. Moreover, help was dependent on at least one factor in addition to TCGF, because a high level of synergy occurred between TCGF and the "help-deficient" 120-hr allogeneic supernatant. In conclusion, the mechanism whereby Lyt-1, Ia- T cells regulated B cell activation with positive and negative allogeneic effects was through the production and subsequent exhaustion of TCGF, respectively. The production of TCGF and help was radioresistant, but exhaustion of TCGF and suppression was radiosensitive.

Animals↗
PubMed1984

Differences in the occurrence of hypertension among (NZB X NZW)F1, MRL-lpr, and BXSB mice with lupus nephritis.

Lupus-prone (NZB X NZW)F1 (B X W) mice and MRL-lpr and BXSB mice were examined for the prevalence of hypertension and levels of plasma renin activity (PRA). Hypertension (greater than 145 mmHg) was observed only in female and male B X W mice with severe nephritis; in female MRL-lpr and male BXSB mice severe nephritis developed without blood pressure elevation (80-135 mmHg). The B X W parental strains, NZB and NZW, and the MRL-lpr congenic partners, MRL- +, did not become hypertensive as they aged. Other strains of mice, aged 3-32 months (A/HeN, BALB/cJ, BALB/cByJ, B10.S/Sg, B10.D2/ oSn , CBA/J, C3H/HeJ, SJL/J and [SJL X NZW]F1), also had normal blood pressure (98-122 mmHg). All mice with lupus nephritis had low PRA, even those with hypertension; furthermore, the MRL-lpr strain had low or undetectable PRA (2 +/- 1 ng/ml/hr), even when kidneys were normal. NZB, NZW, and MRL- + mice had normal PRA (10-16 ng/ml/hr). Thus, B X W mice frequently developed low renin hypertension during the last phase of their renal disease; whereas MRL-lpr and BXSB mice died from renal disease without observable increases in blood pressure.

Animals↗
PubMed1982

Cells transformed by Rous sarcoma virus release transforming growth factors.

Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage-independent colony formation in gelified medium, at the restrictive temperature (41 degrees 5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.

Animals↗
PubMed1982

Differential uptake of 2-fluoro-2-deoxyglucose by normal and transformed chicken and mouse fibroblasts as a function of glucose concentration.

Recently, [18F]-2-fluorodeoxyglucose, a positron emitter, has found an important application in certain metabolic studies using external scanning techniques. We have studied [14C]-2-fluorodeoxyglucose, [3H]-2-deoxyglucose and [14C]-D-glucose uptake in normal and transformed chicken and mouse fibroblasts to determine whether such external scanning techniques might be applicable to detect in vivo tumoral localisations. In this in vitro model, with glucose concentrations in the culture medium below that of normal animal blood, transformed cells showed a higher uptake of all three hexose tracers than did normal cells. However, at physiological glucose concentrations there was no differential uptake of these hexoses. Therefore our results suggest that [18F]-fluorodeoxy-glucose would be of no help in the in vivo detection of tumour localisations by external positron scanning unless in vitro and in vivo cell behavior differs considerably.

Animals↗