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Biomedical subjects

G C Douglas

Publications and source records attributed to G C Douglas.

At least 37 records · Page 2Linked to original sources
PubMed1994

Effect of cytokines and anti-adhesion molecule antibodies on the adhesion of lymphocytic cells to human syncytiotrophoblast.

We have previously shown that lymphocytic cells bind to cultured syncytiotrophoblast and that this may be important in the lymphocyte-mediated infection of trophoblast with the human immunodeficiency virus (HIV). Leukocyte-trophoblast adhesion may also have implications for normal trophoblast function. The following experiments were designed to characterize the adhesion systems that mediate the attachment of lymphocytic cells to trophoblast. Adhesion was assayed by labelling lymphocytic MOLT-4, clone 8 cells with the fluorescent marker, calcein-AM, and then incubating them with primary cultures of human syncytiotrophoblast. Adhesion was stimulated by pretreatment of the trophoblast cultures with several cytokines either alone or together. These included tumor necrosis factor-alpha (TNF-alpha), granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). Stimulation was time- and dose-dependent. In contrast, preincubation of trophoblast cultures with anti-TNF-alpha antibodies for 2 days reduced MOLT adhesion by almost 50%. Preincubation with other anti-cytokine antibodies had no significant effect on adhesion. In other experiments, adhesion was measured in the presence of antibodies to known adhesion molecules. Adhesion was reduced by 50% in the presence of antibodies to alpha 4 integrin or beta 1 integrin. When present together, these antibodies reduced adhesion by almost 85%. Incubation in the presence of antibodies to the very late activation antigen-4 (VLA-4; alpha 4 beta 1 integrin) counter-receptors, VCAM-1 and CS-1, was without effect. Adhesion was also unaffected by antibodies to LFA-1, ICAM-1, ICAM-2, LFA-2, or LFA-3. These results suggest that adhesion is mediated by an adhesion system consisting of lymphocyte VLA-4 (alpha 4 beta 1) and an as yet unidentified counter receptor on trophoblast.

Antibodies↗
PubMed1994

Binding of insulin-like growth factor I to human trophoblast cells during differentiation in vitro.

The binding of insulin-like growth factor I (IGF-I) to its receptor stimulates cell proliferation and differentiation in several cell systems. In the present studies, we have characterized the expression of IGF-I receptors during trophoblast differentiation in vitro. During differentiation of cytotrophoblast cells to syncytiotrophoblasts, IGF-I binding capacity increased 40 per cent from 80 to 130 fmol/mg protein on day 1 to day 2, then decreased by 70 per cent from 130 to 40 fmol/mg protein on day 2 to day 3. IGF-I binding affinity increased non-significantly with a change in kDa from 6.5 to 3.5 nM between day 1 and day 3 in culture. In contrast, when cells were maintained in an undifferentiated state, there was no change in either binding capacity or affinity over three days in culture. Down-regulation of the IGF-I receptor by pre-incubation with IGF-I was demonstrated for syncytiotrophoblasts with a maximum decrease in IGF-I binding of 67 per cent after 72 h pre-incubation. Minimal down-regulation was seen for the cytotrophoblast cells (maximum decrease in IGF-I binding of 22 per cent after 48 h pre-incubation). In conclusion, these results demonstrated that IGF-I binding to the trophoblast cell surface changes during trophoblast differentiation in vitro and suggest involvement of IGF-I in this process.

Cell Differentiation↗
PubMed1994

Induction and stability of somaclonal variation in growth, leaf phenotype and gas exchange characteristics of poplar regenerated from callus culture.

Populus trichocarpa Torr. and Grey x P. balsamifera L. TT32 lines were regenerated from calli that had been maintained under differing in vitro conditions for sixteen months. In the final months, calli were maintained with one of six concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D, 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6 mg l(-1))and regenerated with 0.25, 0.50 or 1.0 mg l(-1) benzylaminopurine (BA). Regenerant lines were obtained from 15 of these 18 treatments. The spectrum of variation in several morphological, physiological and leaf gas exchange traits was evaluated in the primary regenerants in 1986, and in their secondary vegetative propagules in the two subsequent years, in relation to differences in the original culture conditions. The results indicate that somoclonal variation was induced largely as a result of prolonged culture in the presence of 2,4-D, but that the terminal maintenance and regeneration phases also induced changes in the regenerants. Qualitative differences among the regenerant lines were detected by the end of 1986. For most traits, these differences were statistically confirmed within the 3-year period. The treatment lines ultimately diverged sufficiently to produce lines showing general performance that was either above or below that of the original TT32 clone. An early visible indicator of this divergence was variation in leaf shape (leaf length/width ratio), which could be related to 2,4-D-BA interactions in the final stages of culture. Graphic illustration of the independent effects of either 2,4-D or BA on stem height and gas exchange parameters suggested an inverse relationship with BA concentration and a complex interaction with 2,4-D. Significant correlations were detected between gas exchange parameters and morphological characteristics representing leaf form and stem development. Overall, the results indicate the presence of somaclonal lines that offer potential for the selective improvement of growth using morphological and gas exchange parameters as screening tools.

Journal Article↗
PubMed1994

Somaclonal variation in growth, leaf phenotype and gas exchange characteristics of poplar: utilization of leaf morphotype analysis as a basis for selection.

Populus hybrid TT32 lines produced from 15 treatment tissue culture regimes exhibited somaclonal variation in morphological and gas exchange parameters. Within four years of regeneration, discrete lines showing statistically validated superior, or inferior, growth performance relative to the parental reference clone were identified. Significant differences in the ratio of leaf length/width between treatment lines provided the earliest reliable indicator of the divergence in overall growth performance. Despite discernible variation in leaf phenotype among primary regenerants and secondary propagules within individual lines, the leaf length/width ratio was identified as a potential parameter for predicting growth performance. Its subsequent use led to the recognition of four distinct leaf morphotypes; cordate, ovate, reniform and oval. Two or more of these morphotypes were distributed within each of the original 15 treatment lines. Regrouping the data on the basis of leaf morphotype resulted in a clear segregation of the morphological traits, and revealed differences that were not readily apparent by statistical analysis based on treatment groups. The demonstration of similar relative performances by individual morphotypes with respect to a range of growth and gas exchange parameters confirmed that variation in leaf morphology was indicative of differential photosynthetic performance. Somaclonal variants with a leaf morphology was indicative of differential photosynthetic performance. Somaclonal variants with a leaf morphotype closest to that of the parental line showed the highest overall potential for selection, suggesting that the greatest benefits accrue from a minimal disturbance of the parental leaf phenotype.

Journal Article↗
PubMed1993

Adhesion of lymphocytic cells to human trophoblast cells in vitro.

The adherence of lymphocytic MOLT-4/clone 8 cells and normal human peripheral blood mononuclear cells (PBMCs) to primary cultures of term human syncytiotrophoblast has been characterized. Adherence was measured using a fluorescence-based assay in which leukocytic cells were labelled with calcein-AM. Adherence of MOLT cells to syncytiotrophoblast increased in a time-dependent fashion up to about 4 h after which adhesion decreased. Adhesion was detectable at 4 degrees C but was greatly reduced compared to that seen at 37 degrees C. Binding increased linearly as the ratio of MOLT cells to trophoblast was increased. Scanning and transmission electron microscopy of MOLT cell-trophoblast cocultures revealed lymphocytes adherent to the free microvillous surface of the syncytiotrophoblast masses. MOLT cells also adhered to cytotrophoblast but the extent of binding was lower than to syncytiotrophoblast. Normal human peripheral blood mononuclear cells adhered to syncytiotrophoblast. Preincubation of trophoblast cells with trypsin in the presence of calcium had no effect on subsequent adhesion of MOLT cells. However, preincubation of trophoblast cells with trypsin in the absence of divalent cations reduced subsequent adhesion. Adhesion of MOLT cells to syncytiotrophoblast was dependent on magnesium and calcium. These results show for the first time that lymphocytic cells adhere to isolated human syncytiotrophoblast and raise the possibility that this may be an important phenomenon in vivo.

Calcium↗
PubMed1993

HEPES buffer perfusate alters rabbit lung endothelial permeability.

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) has been shown to cause changes in cultured endothelial cells and smooth muscle function at concentrations from 5 to 25 mM. To determine whether HEPES also affects vascular permeability, the effects of two buffers, HEPES and phosphate, were compared in isolated perfused rabbit lungs. Hemodynamic parameters and vascular protein permeability-surface area products (PS) were measured after perfusion with the buffers. Endothelial permeability was measured for an anionic and a cationic albumin to assess the charge effects of the zwitterion buffer. With HEPES, there were no changes in vascular pressure or resistance but permeability was affected. Cationic albumin permeability increased with 12 mM HEPES (8.7phosphate-->30(12) mM HEPES x ml.min-1.g dry lung-1 x 10(-2)) as did the anionic albumin PS (2.7phosphate-->3.52(12) mM HEPES). The cationic PS returned to baseline (8.1(60) mM HEPES) at 60 mM HEPES, but the anionic PS did not change from the 12 mM HEPES (4.01(60) mM HEPES). In summary, we find that HEPES is not innocuous. Although hemodynamic parameters did not change, endothelial permeability was increased when HEPES was used at normal concentrations. Therefore, HEPES should be used with caution as a physiological buffer in perfused organ systems.

Animals↗
PubMed1992

Maternal-fetal transmission of human immunodeficiency virus: a review of possible routes and cellular mechanisms of infection.

The prevalence of human immunodeficiency virus (HIV) infections among children is increasing in a manner that closely follows the spread of the disease among women. Despite the fact that in utero transmission via the placenta is though to play a major role in the spread of HIV to the pediatric population, little is known about the timing, route(s), and cellular mechanisms by which maternal-fetal transmission occurs. This review attempts to use a developmental and cellular approach to assess the available clinical and laboratory data pertaining to maternal-fetal HIV transmission. While much of this review focuses on the role of the placenta, particularly the placental trophoblast, on the transmission of HIV, potential routes of infection during early development are also discussed. Clinical studies indicate that the placental trophoblast can be infected with HIV but have shed no light on how the virus gains entry to this tissue. While some laboratory studies confirm that trophoblast cells and placental macrophages can be infected with HIV in vitro, many studies are difficult to interpret because of inadequate characterization of the placental cells used. The role of CD4 in the infection of trophoblast remains controversial and clearly warrants a systematic examination. It is also apparent that viral tropism has not received enough attention and more studies using different strains of HIV are required. Thus, several basic questions remain to be answered before strategies to prevent maternal-fetal transmission of HIV can be developed.

Female↗
PubMed1991

Cell-mediated infection of human placental trophoblast with HIV in vitro.

In order to investigate how human immunodeficiency virus (HIV) gains entry to the placenta, we have performed in vitro experiments in which highly purified trophoblast cells isolated from term human placentas were examined for their susceptibility to HIV infection. Trophoblast cells were exposed to cell-free HIV-1 for up to 24 h, after which the cultures were monitored by p24 antigen capture assay, reverse transcriptase assay, and electron microscopy for evidence of virus uptake and replication. None was found. In the second series of experiments, trophoblast cells were cocultured with HIV-infected MOLT-4 cells for 24 h, stained using an anti-HIV antibody, and examined by immunofluorescence microscopy. The MOLT cells were strongly positive, as expected, but many trophoblast colonies also showed a punctate staining pattern. Examination of similar cultures using the electron microscope revealed MOLT cells adherent to trophoblast but no evidence of cell-cell fusion. Virions were observed in coated pits at the trophoblast cell surface and in endosomes or multivesicular bodies in the cytoplasm. These observations are consistent with an endocytosis-mediated mechanism of virus entry. Virions were also observed budding from the trophoblast plasma membrane, indicating that these cells can support HIV replication. To our knowledge, these results show for the first time that HIV can infect placental trophoblast cells in vitro. The results suggest that the placenta could become infected with HIV by the interaction of virus-infected maternal lymphocytes with syncytiotrophoblast bordering the maternal blood in the intervillous space.

Female↗
PubMed1990

Isolation and morphologic differentiation in vitro of villous cytotrophoblast cells from rhesus monkey placenta.

Trophoblast is the major functional cell type of the placenta. The purpose of this study was to devise a means to isolate trophoblast cells from the monkey placenta and to examine their capacity to differentiate in vitro. Methods originally devised for the isolation of human cytotrophoblast cells produced poor yields and a low degree of purity when applied to the near-term rhesus monkey placenta. However, a procedure has been developed which allows the isolation of a cell population consisting of more than 95% cytotrophoblast based on intermediate filament immunocytochemistry. The cells sedimented between densities of 1.040 and 1.053 g/ml on continuous Percoll density gradient centrifugation. When maintained in culture the cells adhered and formed aggregates of mononuclear cells by 24 h. By 5 d in culture, immunofluorescent staining using antidesmoplakin and antinuclear antibodies revealed that most colonies consisted of large multinucleated masses similar to syncytiotrophoblast. These results demonstrate trophoblast cells from monkey placental villi can be isolated with a high degree of purity and undergo morphologic differentiation in vitro. This preparation should enable investigators to study many functional characteristics of these cells throughout gestation.

Animals↗
PubMed1990

Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei.

The differentiation of isolated human cytotrophoblast cells has been studied by staining cells with anti-desmoplakin antibodies, to reveal cell boundaries, and with anti-nuclear antibodies, to reveal nuclei. During the first 24 h after plating in Ham's/Waymouth medium, mononucleated cytotrophoblast cells began to spread and aggregate, forming colonies. This was accompanied by progressive changes in the pattern of desmoplakin staining. In single cells, desmoplakin was dispersed throughout the cytoplasm. As cells aggregated, desmoplakin was redistributed and formed linear, punctate arrays at regions of cell-cell contact, consistent with desmosome formation. A pavement-like staining pattern was maintained even at 5 days. Double staining for desmoplakin and nuclei revealed that most cells within colonies were mononucleated. When plated in a growth medium originally formulated for keratinocytes, cytotrophoblast cells aggregated and formed desmosomes normally. However, after 48 h, cell diameters were increased and nuclei changed from being evenly distributed to forming clusters within large cells, consistent with syncytiotrophoblast formation. While cells grown in Ham's/Waymouth medium for 2 days could be induced to differentiate by switching to keratinocyte growth medium, cells cultured for 5 days before switching were resistant to the differentiation-inducing effects of the keratinocyte medium. Desmosome-type junctions within colonies of trophoblast cells were unstable and, even after 5 days in culture, could be disrupted by lowering the extracellular Ca2+ concentration. While syncytiotrophoblast formation in keratinocyte growth medium (which contains epidermal growth factor, insulin and hydrocortisone) was accompanied by a 15- to 20-fold increase in chorionic gonadotropin secretion, syncytiotrophoblast formation occurred to a similar extent in keratinocyte basal medium (which does not contain these factors) but with only a twofold increase in chorionic gonadotropin release. These results support the notion that biochemical and morphological differentiation of trophoblast are independent events.

Antibodies, Monoclonal↗
PubMed1989

Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres.

A procedure has been developed which yields a pure population of villous cytotrophoblast from term human placenta. As a first step, a cell preparation highly enriched for cytotrophoblast (identified by positive cytokeratin staining) was obtained using a modification of the method of Kliman et al. (1986). The remaining contaminating cells (identified by positive vimentin staining) were then removed by treatment with mouse monoclonal antibodies against class I and class II major histocompatibility antigens followed by magnetic microspheres coated with goat anti-mouse IgG. The rationale for this step was based on the fact that villous trophoblast fails to express HLA antigens whereas cells from the villous mesenchyme do express these surface antigens. Rosetted cells were immobilized using a magnet allowing the non-rosetted cells to be easily withdrawn by pipette. When the non-rosetted cells were placed in primary culture, no HLA-positive or vimentin-positive cells could be detected using immunofluorescence microscopy, indicating complete removal of these components by the immunomagnetic separation procedure. The cells were positive for cytokeratin and, after 24 h, showed positive staining for pregnancy-specific beta 1-glycoprotein (SP1) and human chorionic gonadotropin. Recovery of cytotrophoblast was greater than 92% with only a slight loss of viability.

Animals↗
PubMed1985

Endocytosis and subsequent processing of 125I-labelled immunoglobulin G by guinea pig yolk sac in vitro.

We have developed conditions for studying the binding, uptake, degradation and transport of 125I-labelled IgG by yolk sac in vitro. Specific binding to tissue at 4 degrees C and to paraformaldehyde-treated tissue at 37 degrees C was time- and temperature-dependent and showed saturation kinetics (Kd,4 degrees C = 2.9 X 10(-6) M, Kd,37 degrees C = 5.3 X 10(-6) M). Uptake was studied at 37 degrees C using untreated tissue (K uptake = 13.3 X 10(-6) M) and was inhibited by preincubation with metabolic poisons but not with cycloheximide. Tissue that had been incubated with 125I-labelled IgG at 37 degrees C released radiolabelled degradation products and intact 125I-labelled IgG into the medium. Experiments with paraformaldehyde-treated and untreated tissue showed that release of intact 125I-labelled IgG was mostly the result of ligand dissociation from surface binding sites. However, more 125I-labelled IgG was released from untreated tissue than could be accounted for solely by loss of surface-bound ligand and the difference was presumed to reflect uptake, transport and exocytosis of 125I-labelled IgG. Degradation of 125I-labelled IgG was inhibited by leupeptin and lysosomotropic amines. These drugs had no detectable effect on 125I-labelled IgG release. The results suggest that degradation and transport of IgG are not intimately related and are consistent with a previously proposed model for IgG transport via coated vesicles which do not fuse with lysosomes and for non-selective uptake into another class of vesicle which does fuse with lysosomes.

Adenosine Triphosphate↗
PubMed1984

A filter paper sandwich method using small volumes of reagents for the detection of antigens electrophoretically transferred onto nitrocellulose.

A method is described which allows antigens electrophoretically 'blotted' onto nitrocellulose to be probed using small volumes (less than 0.3 ml) of antibody solutions. Reagent volumes required for this method are more than 10-fold lower than the minimum volumes attainable using either custom made or commercial incubation trays. The procedure is simple and economical and should be applicable to any situation where it is desirable to either conserve or reduce the amount of screening reagent used.

Antibodies, Monoclonal↗
PubMed1981

Lysosomal enzyme secretion from human placental villi is increased by serum albumin and IgG.

Human serum albumin and IgG increased the secretion of the lysosomal enzyme, beta-hexosaminidase, from human placental villi in a dose- and time-dependent manner. Release was also enhanced by bovine serum albumin and ovalbumin. beta-Hexosaminidase secretion was not increased by the non-endocytosable substrate, IgG-Sepharose, indicating that the signal for release is not given by the interaction of protein with the cell surface. Experiments with 125I-labelled albumin and IgG showed that both proteins were actively internalised by placental villi giving credence to the idea that secretion is triggered after uptake.

Chorionic Villi↗
PubMed1980

Secretion of lysosomal enzymes by human placental villi in vitro.

Chorionic villi from first trimester and term human placentas have been incubated in vitro and shown to release the lysosomal enzymes, beta-hexosaminidase, alpha-glucosidase and beta-glucuronidase. There was negligible release of the cytoplasmic enzyme, lactate dehydrogenase, under the same conditions. The first trimester villi released proportionally more of their lysosomal enzyme content than did the term villi. Extracellular levels of beta-hexosaminidase were raised and those of alpha-glucosidase and beta-glucuronidase were lowered when tissue was incubated with 1 microM colchicine, suggesting that microtubules are involved in the control of lysosomal enzyme release from placental villi.

Chorionic Villi↗
PubMedDate not supplied

Receptor-mediated endocytosis of 125I-labelled transferrin by human choriocarcinoma (JAR) cells.

The binding and subsequent internalization of 125I-labelled transferrin has been studied in the human choriocarcinoma cell line, JAR. At 4 degrees C, binding was time- and concentration-dependent and exhibited saturation kinetics. The results indicate 1.6 x 10(6) binding sites per cell surface with a Kd of 4.12 x 10(-9) M. Experiments with saponin-permeabilized cells revealed a large intracellular receptor pool constituting 72 per cent of the total cell receptor population. Evidence of ligand internalization at 37 degrees C was obtained by showing the progressive resistance of cell-associated radioactivity to acid treatment. Pulse-chase experiments showed that after internalization, ligand resided within the cell for about 6 min before being released back to the medium in an intact form. Ligand release, but not internalization, was almost completely inhibited by exposure to monensin. Preincubation of cells at 37 degrees C in the presence of monensin and under transferrin-free conditions had no effect on the subsequent binding of 125I-transferrin. Pulse-chase experiments using 125I-labelled anti-transferrin receptor antibody suggest that receptors are internalized in the absence of added transferrin with a rate coefficient of 0.036 min-1. In the presence of unlabelled transferrin, the initial rate of labelled antibody internalization was increased (rate coefficient was 0.23 min-1) but the maximal amount of antibody internalized remained virtually unchanged. Internalized antibody accumulated intracellularly with no evidence of release or degradation.

Antibodies, Monoclonal↗
PubMedDate not supplied

Effects of monensin on the receptor-mediated endocytosis of 125I-labelled IgG by guinea-pig yolk sac in vitro.

The effects of the carboxylic ionophore, monensin, on the receptor-mediated binding and uptake of 125I-labelled IgG by the guinea-pig yolk sac have been studied in vitro. Exposure of tissue to 10 microM monensin resulted in a rapid inhibition of uptake which correlated with a time- and temperature-dependent loss of cell-surface receptor activity. Monensin appeared to bring about a change in receptor distribution since the lost activity could be detected after permeabilizing the tissue with saponin. Electron microscopic examination of monensin-treated tissue revealed that the apical plasma membrane of endoderm cells was depleted of coated and uncoated pits and that the apical cytoplasm contained numerous large vacuoles. Dilation of the Golgi apparatus was also observed. Normal surface receptor activity and ultrastructural features could be largely recovered by removal of monensin. Recovery of receptor activity was unaffected by the presence of cycloheximide. These results are consistent with a model in which IgG receptors are recycled and in which monensin blocks this process by causing receptors to be trapped intracellularly. Ammonium chloride or a combination of valinomycin and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone also brought about a loss of surface IgG receptors, lending support to the idea that inhibition of recycling was the result of perturbation of an intracellular acidification event and implying that passage through an acidic compartment may be important for correct receptor processing.

Animals↗