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S K Smith

Publications and source records attributed to S K Smith.

At least 127 records · Page 7Linked to original sources
PubMed1995

Localization and quantification of vascular endothelial growth factor messenger ribonucleic acid in human myometrium and leiomyomata.

Vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and VEGF peptide were studied in human myometrium and leiomyomata. Amplification of complementary DNA from myometrium and leiomyomata by the polymerase chain reaction revealed three species of mRNA encoding VEGF: VEGF189, VEGF165, and VEGF121. In situ hybridization demonstrated VEGF mRNA expression throughout the smooth muscle cells of myometrium and leiomyomata. VEGF-like immunoreactivity was also detected in these tissues by immunocytochemistry. Quantification of VEGF mRNA using an RNAse protection assay demonstrated that in normal myometrium, levels of VEGF mRNA are significantly higher in the secretory phase than in the proliferative phase of the cycle. Leiomyomata did not have significantly different levels of VEGF mRNA compared with normal myometrium. In untreated leiomyomata, there was no significant difference between VEGF mRNA levels in the proliferative and secretory phases of the cycle. Leiomyomata from women treated with a GnRH anlog did not have significantly different levels of VEGF mRNA from untreated leiomyomata.

Adult↗
PubMed1995

Ultrasonographic growth measurements in triplet pregnancies.

The incidence of triplet pregnancies is increasing due to the widespread use of ovulation induction agents and assisted conception treatments. The aim of this study was to acertain the normal ultrasonographic measurements for fetal growth parameters in triplet pregnancies. The ultrasonographic measurements of all triplet pregnancies managed in two major hospital centres were reviewed retrospectively and those in which there was less than 25% discordance in birth weight were included in the study. Triplet 50th centile for fetal biparietal diameter, whilst falling through the normal singleton centile range from 27 weeks gestation, did not fall below the 10th centile value of singletons. Triplet 50th centile for head circumference was equivalent to the singleton 10th centile from 23 weeks gestation. Triplet 50th centile for abdominal curcumference was persistently below that of the singleton 50th centile, falling below the singleton 10th centile from 29 weeks gestation. Normal growth rate of triplet gestations in the third trimester of pregnancy varies from that of singletons. An awareness of this altered growth rate is necessary to avoid the inappropriate diagnosis of asymmetrical triplet fetal growth retardation. We suggest that the growth curves presented here may be used to monitor triplet fetal growth.

Birth Weight↗
PubMed1995

Localization of vascular endothelial growth factor and its receptor, flt, in human placenta and decidua by immunohistochemistry.

Vascular endothelial growth factor is a secreted angiogenic growth factor the mRNA of which is present in the placenta. The mRNA encoding the vascular endothelial growth factor receptor, flt, has also been demonstrated in placenta, with trophoblast appearing to be a novel site of flt expression. We investigated the expression of both vascular endothelial growth factor and flt-like immunoreactivity in first trimester and term placentae. In the first trimester, vascular endothelial growth factor immunoreactivity was localized to placental macrophages (Hofbauer cells), and in decidua, to glandular epithelium and maternal macrophages. In the term placenta, vascular endothelial growth factor immunoreactivity was present in extravillous trophoblast and in extracellular material. Flt immunoreactivity was demonstrated on extravillous trophoblast in first trimester and term, and on Hofbauer cells within placental villi. This complex pattern of both vascular endothelial growth factor and flt-like immunoreactivity suggests that vascular endothelial growth factor may be involved not only in the regulation of placental angiogenesis, but also in trophoblast invasion.

Decidua↗
PubMed1994

Screening for cytokine messenger ribonucleic acids in purified human decidual lymphocyte populations by the reverse-transcriptase polymerase chain reaction.

At the time of human embryo implantation, large numbers of maternal CD56brightCD16- NK cells appear in the uterus. These unusual lymphocytes are believed to control the migration and differentiation of highly invasive fetally derived trophoblast cells, which infiltrate into the maternal uterus to remodel the spiral arteries during the first trimester. One possible mechanism of control is by cytokine production. In this study, highly purified (> 99%) populations of first trimester decidual CD56brightCD16- NK cells and CD3+ T lymphocytes were obtained by using a FACS. These cells were examined by reverse transcriptase PCR for their expression of mRNAs for the following cytokines: granulocyte-macrophage (GM)-CSF, CSF-1, TNF-alpha, IFN-gamma, TGF-beta 1, leukemia-inhibitory factor (LIF), and IL-2. Then, the expression was compared with that of resting PBL. The identity of the PCR products was verified by Southern blotting and hybridization with cytokine-specific probes. Both decidual CD56brightCD16- NK cells and CD3+ T cells were found to express mRNA for CSF-1, TNF-alpha, IFN-gamma TGF-beta 1, and LIF, but GM-CSF mRNA was detected only in CD56bright NK cells. IL-2 mRNA was detected in only some decidual T cell samples, and then only after at least two rounds of amplification. In contrast, peripheral blood CD56brightCD16- NK cells, CD56dimCD16+ NK cells, and CD3+ T cells expressed mRNA only for TNF-alpha and TGF-beta 1, but not for GM-CSF, CSF-1, IFN-gamma, LIF, or IL-2. These results suggest that both decidual NK cells and decidual T cells produce a variety of cytokines that may be involved in the control of trophoblast migration and differentiation during pregnancy.

Antigens, CD↗
PubMed1994

Quantification of mRNA in human tissue using fluorescent nested reverse-transcriptase polymerase chain reaction.

We report the development of a quantitative nested reverse-transcriptase polymerase chain reaction which utilizes a fluorescence detection system. Using specific primer pairs to study mRNA for endothelin receptors in the human kidney, we synthesized a cRNA construct containing the same sequences but yielding a PCR product some 300 base pairs larger than native mRNA. Inclusion of a known amount of construct as internal standard with tissue RNA prior to cDNA synthesis allowed all reactions to occur under the same conditions in the same tube. In the nested PCR reaction, serial dilutions made before the second round enabled construction of a standard curve for each assay, and confirmation that standard and sample curves remained parallel. This indicates that both cDNAs amplified at the same rate. One internal primer was fluorescently labeled. Quantification of products using an ABI 373A sequencer with Genescan software gave sensitive and reproducible results. Analysis of a needle biopsy (10 mg) of histologically normal cortex gave 0.4 amol ETA mRNA and 1.6 amol ETB mRNA/micrograms total RNA. In medulla these values were 0.46 and 1.16 amol/micrograms, respectively. Ratios of ETB to ETA message were 74:26 in cortex and 77:23 in medulla, agreeing with previous ligand binding studies of receptor protein. Intra- and interassay coefficients of variation were 4.5 and 5.3%. This new method has potential for widespread application to the study of low copy-number mRNA or where only very small amounts of tissue are available, such as biopsy specimens.

Base Sequence↗
PubMed1994

Tissue collagenase: serum levels during pregnancy and parturition.

Serum levels of tissue collagenase, matrix metalloproteinase-1, were measured in both longitudinal and cross-sectional studies, in 332 pregnant women and 27 non-pregnant volunteers. The enzyme-linked immunosorbent assay (ELISA) used is the first described to measure collagenase in serum directly, is specific, and is rapid and reproducible. Levels were determined throughout pregnancy, during term and preterm labour, and in the post-partum period. Serum tissue collagenase levels were elevated in pregnancy (P < 0.001). There was no difference between levels of serum collagenase prior to labour at term and those observed during labour. Similarly, there was no significant difference in levels obtained during preterm labour and those at a similar gestation in women who subsequently delivered at term. No significant decrease in levels had occurred by the 4th post-partum day. In view of these findings of unaltered matrix metalloproteinase-1 levels in association with labour, previous reports of raised serum collagenase activity in association with the onset of spontaneous labour, at term and preterm gestation periods, may be due to increased neutrophil collagenase activity.

Collagenases↗
PubMed1994

Expression of c-kit and kit ligand at the human maternofetal interface.

Kit ligand, or stem cell factor, is a recently identified growth factor, which binds to and activates the c-kit proto-oncogene, and which has been shown to act synergistically with other haematopoietic growth factors in the bone marrow. We have previously shown that several isoforms of kit ligand, which arise due to alternative splicing, are expressed in human placenta. In order to elucidate the role of c-kit and its ligand during human placental development we have investigated the expression of c-kit and kit ligand in human first trimester and term placenta as well as in pregnant and non-pregnant endometrium, by immunocytochemistry and flow cytometric analysis. In non-pregnant endometrium no expression of kit ligand was seen. By contrast, in first trimester decidua, kit ligand was strongly expressed by the arterial media of maternal blood vessels. Kit ligand was also expressed throughout pregnancy by invasive fetal extravillous trophoblast, and by fetal fibroblasts within the placental villi. c-kit was found to be expressed on Hofbauer cells within the chorionic villi, and by decidual macrophages at all stages in pregnancy. c-kit was also detected on the small CD56dim subset of uterine large granular lymphocytes which form the major leukocyte population in human first trimester decidua. Our results suggest that kit ligand may be involved in the regulation of fetal macrophages, and in particular in signalling between invading extravillous trophoblast which expresses kit ligand, and maternal leukocytes bearing the c-kit receptor.

Amino Acid Sequence↗
PubMed1994

Vascular endothelial growth factor receptor localization and activation in human trophoblast and choriocarcinoma cells.

Vascular endothelial growth factor (VEGF; also known as vascular permeability factor) is a secreted angiogenic growth factor. It is highly specific for endothelial cells, and its receptor, the fms-like tyrosine kinase (flt), has been localized only to endothelial cells in vivo. Here we describe the expression of mRNA encoding flt in human trophoblast as revealed by in situ hybridization. This mRNA is highly expressed in the cytotrophoblast shell and columns and also highly expressed by the extravillous trophoblast (EVT) in the maternal decidua both in the first trimester and at term. The trophoblast-like choriocarcinoma cell line BeWo also expresses this receptor and the related receptor, kinase domain-containing receptor (KDR), which is also a receptor for VEGF. Treatment of the cell line BeWo with VEGF165 stimulated 3H-thymidine incorporation and tyrosine phosphorylation of MAP (mitogen-activated protein) kinase in a time- and dose-dependent fashion. This study is the first demonstration of the presence of flt on non-endothelial cells in vivo and suggests a role for VEGF in the growth and differentiation of cytotrophoblast at implantation.

Base Sequence↗
PubMed1994

Tissue inhibitor of metalloproteinases: serum levels during pregnancy and labor, term and preterm.

OBJECTIVE: To determine the levels in serum of tissue inhibitor of metalloproteinases (TIMP) in pregnancy and to examine the possibility of a time course in relation to parturition, both term and preterm. METHODS: Serum tissue inhibitor of metalloproteinases was measured using an enzyme-linked immunosorbent assay. A cross-sectional study was conducted in 333 women during pregnancy, labor, and the postpartum period and in 27 nonpregnant volunteers. Longitudinal data were obtained from 22 women who provided a sample at term, during labor, and in the postpartum period. RESULTS: In uncomplicated pregnancies, serum TIMP levels were low from the onset of pregnancy until 37 weeks' gestation, in comparison to levels in nonpregnant women (P < .001). During the final weeks of pregnancy, levels rose and at 37-42 weeks were similar to nonpregnant levels. The levels did not change with the onset of labor. Serum concentrations of TIMP obtained during preterm labor were elevated compared to a control group of patients at a similar gestation who subsequently delivered at term (P < .01). Serum TIMP levels were significantly higher during the postpartum period than at all other times (P < .001). CONCLUSIONS: Changes in serum TIMP levels during and after pregnancy may parallel the remodeling of the extracellular matrix that takes place throughout this period. Further work is necessary to evaluate the prognostic value of TIMP for preterm labor.

Cross-Sectional Studies↗
PubMed1994

Protection studies with integral membrane fractions of Haemonchus contortus.

Techniques for targetting glycoproteins integral to the luminal membrane of the intestinal cells of Haemonchus contortus were used to isolate fractions of whole parasites with protective antigen potential. Sheep immunization trials with various candidate fractions revealed one which selectively bound to lectins with specificity for N-acetylgalactosamine and which reduced mean challenge worm burdens by up to 72% and mean faecal egg counts by up to 93%. The luminal surface of the intestines of the Haemonchus recovered from sheep immunized with this antigen were coated with host immunoglobulin, suggesting that the protective effect was due to antibodies interfering with the function of the gut. Further biochemical characterization of this fraction, which has been termed Haemonchus galactose-containing glycoprotein complex (H-gal-GP complex), showed that it could be distinguished from previously described protective antigens of this parasite and that it was only detectable in detergent extracts of the worms.

Acetylgalactosamine↗
PubMed1994

Basic FGF activates phospholipase D in endothelial cells in the absence of inositol-lipid hydrolysis.

In the absence of inositol-lipid hydrolysis, mitogenic concentrations of basic fibroblast growth factor (bFGF) stimulated phosphatidylbutanol formation in the presence of butan-1-ol in [3H]myristate-labeled human umbilical vascular endothelial (HUVE) cells, indicating that the fibroblast growth factor receptor was able to couple to the activation of phospholipase D (PLD). The ability of bFGF and 12-O-tetradecanoylphorbol-13-acetate (TPA) to stimulate PLD activity was completely abolished in cells pretreated with 400 nM TPA for 48 h to downregulate protein kinase C (PKC). bFGF-stimulated PLD activity was inhibited by genistein (5 microM; P < 0.02) and the PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 5 microM; P < 0.001) as well as by the removal of calcium from extracellular environment. bFGF induced DNA synthesis in a dose-dependent manner, and pretreatment of cells with H-7 inhibited the mitogenic activity of bFGF. These results indicate that activation of PKC is responsible for bFGF-induced PLD activation and the mitogenic activity of bFGF in HUVE cells. A coupled PLD/3-sn-phosphatidate phosphohydrolase pathway may play a role in the regulation of endothelial cell proliferation.

Calcium↗
PubMed1994

Quantification of messenger ribonucleic acid for epidermal growth factor in human myometrium and leiomyomata using reverse transcriptase polymerase chain reaction.

Epidermal growth factor (EGF) mRNA was quantified in samples of human myometrium, untreated leiomyomata, and leiomyomata from patients treated with a GnRH analog. Quantitative reverse transcriptase-polymerase chain reaction, using a synthetic internal standard, was applied to determine levels of EGF mRNA. In myometrium from uteri with no leiomyomata, levels of EGF mRNA did not differ between the proliferative and secretory phase of the cycle. Leiomyomata from women who had received no drug therapy had significantly higher amounts of EGF mRNA than myometrium from a normal uterus, but only in the secretory phase of the cycle. In the proliferative phase, leiomyomata did not have different amounts of EGF mRNA compared to normal myometrium. Untreated leiomyomata in the secretory phase of the cycle, but not those in the proliferative phase, had significantly more EGF mRNA than leiomyomata from women who had received treatment with a GnRH analog. These findings suggest that EGF is important in leiomyomata development, but imply that its production is only increased during the secretory phase of the cycle. This challenges the hypothesis that EGF production in leiomyomata is mediated by estrogen and raises the possibility that progesterone may be the more important hormone in fibroid growth.

Adult↗
PubMed1994

Leukaemia inhibitory factor mRNA concentration peaks in human endometrium at the time of implantation and the blastocyst contains mRNA for the receptor at this time.

Maternal endometrial leukaemia inhibitory factor (LIF) is required for successful implantation in mice. Mice with homozygous deletions in this gene fail to support implantation. The localization of immunoreactive LIF and the concentration of the mRNA encoding human LIF in normal endometrium during the menstrual cycle were investigated. The amount of RNA was low or undetectable in the proliferative phase but increased by approximately six times in the mid- to late secretory phase. The protein can only be detected by immunocytochemistry in glandular epithelium in the mid- or late secretory phase. To investigate the possible target for the endometrial LIF, we undertook reverse transcription-PCR analysis of early human embryos to determine whether they contain the mRNA encoding the LIF receptor. This study indicated that at the time of implantation in humans, the maternal endometrium produces LIF and that the blastocyst expresses LIF receptor mRNA and therefore may be capable of responding to this signal.

Base Sequence↗
PubMed1994

Functional platelet-activating factor receptors linked to inositol lipid hydrolysis, calcium mobilization and tyrosine kinase activity in the human endometrial HEC-1B cell line.

Platelet-activating factor (PAF; sn-1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine) is thought to be an important mediator of embryo-endometrial interactions in early pregnancy, and an understanding of its role in the establishment of early human pregnancy can only follow an understanding of its mechanism of action. In a human endometrial epithelial cell line, HEC-1B, the presence of mRNA encoding the platelet-activating factor receptor was demonstrated by reverse transcription-polymerase chain reaction. The presence of functional receptors was shown by inositol trisphosphate accumulation and a rise in the concentration of intracellular free calcium evoked by platelet-activating factor in myo-[2-3H]inositol-labelled and fura-2-loaded cells, respectively. Platelet-activating factor evoked rapid and concentration-dependent increases in the concentration of intracellular free calcium and inositol trisphosphate that were inhibited by the platelet-activating factor receptor antagonist WEB 2086, indicating that the responses are receptor mediated. Inositol trisphosphate accumulation evoked by platelet-activating factor was unaffected by pretreatment with pertussis toxin. Platelet-activating factor also stimulated the tyrosine phosphorylation of at least two major proteins of 80 kDa and 44 kDa; the smaller protein is an isoform of mitogen-activated protein kinase. These results show that functional platelet-activating factor receptors are located on the endometrial epithelial cell line HEC-1B and are linked to inositol lipid hydrolysis, calcium mobilization and tyrosine kinase activity.

Azepines↗
PubMed1994

Colocalization of acidic and basic fibroblast growth factor (FGF) in human placenta and the cellular effects of bFGF in trophoblast cell line JEG-3.

The placenta undergoes extensive angiogenesis and cellular proliferation to establish adequate blood supply to the fetus. The aim of this study was to compare and contrast the immunolocalization of acidic and basic fibroblast growth factor (FGF) in both first trimester and term placenta and gestational decidua. Human choriocarcinoma cell line JEG-3 were employed as a model of cytotrophoblast and the effect of basic FGF on cell proliferation and phospholipase C and D activation investigated. Basic FGF-immunoreactivity (IR) was detected in or around cytotrophoblast cells and in extravillous trophoblast in first trimester placenta by immunohistochemistry using primary polyclonal rabbit antibodies. Identical staining patterns were produced by acidic FGF antibodies indicating colocalization of acidic FGF and basic FGF. At term, weaker and more diffuse staining was seen in the syncytiotrophoblast surrounding the placenta villi and strong staining was present in the smooth muscle cells of mid and large size placental vessels and in some endothelial cells. Endothelial cells and extravillous trophoblast stained strongly within the decidua at first trimester, whereas the glandular epithelium was weakly stained. Basic FGF induced [3H]thymidine incorporation in JEG-3 cells in a dose dependent manner and caused an increase in inosital phosphate accumulation in cells pre-labelled with myo-[3H]inosital at similar concentrations, suggesting a role of phospholipase C in JEG-3 cell proliferation. However, basic FGF failed to stimulate phospholipase D activity in cells pre-labelled with [3H]myristic acid. The detection of acid FGF and basic FGF on both maternal and fetal side of the placenta during early pregnancy suggests a role for FGF in angiogenesis, whereas localisation of the growth factor at term, when extensive angiogenesis has diminished, would indicate that FGF may be associated with more differentiated functions of the trophoblast. The nuclear localization of basic FGF in dividing but not non-dividing placental cells together with the effect of basic FGF on JEF-3 cells, strongly supports a role for basic FGF in cytotrophoblast proliferation in vivo.

Cell Division↗