Biomedical subjects

S K Smith

Publications and source records attributed to S K Smith.

At least 55 records · Page 3Linked to original sources

PubMed2000

The effect of progestins on vascular endothelial growth factor, oestrogen receptor and progesterone receptor immunoreactivity and endothelial cell density in human endometrium.

One common side-effect of contraceptive use is that it often leads to disrupted endometrial bleeding patterns. This may be due to changes in endothelial density and vessel integrity. To investigate whether the level of endometrial immunoreactive vascular endothelial growth factor (VEGF), oestrogen receptor or progesterone receptor (PR) have any role in this, women were treated with either Mircette, a monophasic oral contraceptive, or Implanon, a long-acting gestagen, and immunohistochemistry performed. In addition a small number of endometria were studied from women treated with levonorgestrel released from an intrauterine coil. During the untreated normal cycle, there was a significant increase in glandular VEGF immunoreactivity and a significant decrease in PR immunoreactivity in the midand late secretory phases compared to the proliferative phase. There was a significant positive correlation between stromal VEGF immunoreactivity and endothelial cell density. This correlation was also apparent during treatment with Implanon, but not with Mircette. Disrupted bleeding patterns were associated with Implanon and to a lesser extent with Mircette. Both contraceptives significantly reduced glandular VEGF immunoreactivity but the intrauterine treatment with levonorgestrel resulted in strong glandular epithelial staining and intense staining of decidualized stromal cells. Implanon significantly increased glandular PR staining, but Mircette significantly reduced stromal PR staining when compared to secretory phase before-treatment biopsies. There were no changes in endothelial cell density or glandular or stromal ER during the normal cycle, or with use of either contraceptive. There was no association of the parameters measured with bleeding patterns or histological category.

Cell Count↗

PubMed2000

Expression of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in conceptus and endometrium during implantation in the rhesus monkey.

The aim of this study was to analyse the expression of transcripts and proteins for vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in different compartments of the early conceptus at primary implantation sites during lacunar (n = 6), early villous (n = 9) and villous placenta (n = 6) stages of gestation in the rhesus monkey. During the lacunar stage, VEGF expression was observed in the cytotrophoblast cells lining the extraembryonic cavity, but these cells did not express PlGF. With further development, cytotrophoblast cells lining villi, forming columns, and constituting anchoring villi, expressed both VEGF and PlGF during early villous and villous placenta stages. In addition, chorion, amnion and villous stromal cells expressed both VEGF and PlGF proteins and mRNA. During the lacunar stage, all epithelial cells in maternal endometrium generally expressed VEGF, while PlGF expression was observed in the plaque epithelium only. As gestation advanced, the expression of VEGF and PlGF from plaque cells decreased, and in surface and glandular epithelium the expression of VEGF increased, while the expression of PlGF remained unaltered. Decidual stromal cells expressed VEGF and PlGF only at low levels during the lacunar stage, while the expression of both increased during the early villous and the villous placenta stages of implantation. It appears from the present study that the expression of VEGF and PlGF are regulated in a temporal and spatial manner during early stages of implantation and that their concerted actions in placental and maternal compartments play a critical role in the evolving pregnancy in the rhesus monkey.

Animals↗

PubMed2000

Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia.

Endometrial growth and repair after menstruation are associated with profound angiogenesis. Abnormalities in these processes result in excessive or unpredictable bleeding patterns and are common in many women. It is therefore important to understand which factors regulate normal endometrial angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays an important role in normal and pathological angiogenesis. In this study we show that expression of VEGF is regulated by hypoxia in human endometrium. Culture in vitro for 24 h under hypoxic conditions resulted in a 2- to 6-fold increase in VEGF secretion by both stromal and epithelial cells isolated from human endometrium. Quantitative RT-PCR was used to measure VEGF messenger ribonucleic acid (mRNA) levels in these cells. After hypoxia, VEGF mRNA levels increased 1.8-fold in stromal cells and 3.4-fold in glandular epithelial cells. The mRNA for each VEGF splice variant increased to an equal extent. The increase in VEGF secretion by stromal and epithelial cells in response to hypoxia was not altered by treatment at the same time with estradiol or progesterone. In situ hybridization of human endometrium during menstruation, when steroid levels are low but the tissue is subject to ischemia, showed strong hybridization to VEGF mRNA in both stromal and glandular cells. These results show that local factors, such as hypoxia, can regulate VEGF expression in the endometrium. This may play an important part in normal endometrial repair after menstruation. The secretion of VEGF by endometrial cells under hypoxic conditions may also be important in the pathogenesis of endometriosis, because it would be predicted to assist revascularization of desquamated endometrial explants when they attach at ectopic sites.

Adult↗

PubMed2000

Changes in tumorigenesis- and angiogenesis-related gene transcript abundance profiles in ovarian cancer detected by tailored high density cDNA arrays.

BACKGROUND: Complementary DNA array analysis of gene expression has a potential application for clinical diagnosis of disease processes. However, accessibility, affordability, reproducibility of results, and management of the data generated remain issues of concern. Use of cDNA arrays tailored for studies of specific pathways, tissues, or disease states may render a cost- and time-effective method to define potential hallmark genotype alterations. MATERIALS AND METHODS: We produced a 332-membered human cDNA array on nylon membranes tailored for studies of angiogenesis and tumorigenesis in reproductive disease. We tested the system for reproducibility using a novel statistical approach for analysis of array data and employed the arrays to investigate gene expression alterations in ovarian cancer. RESULTS: Intra-assay analysis and removal of agreement outliers was shown to be a critical step prior to interpretation of cDNA array data. The system revealed highly reproducible results, with intermembrane coefficient of reproducibility of +/- 0.98. Comparison of placental and ovarian sample data confirmed expected differences in angiogenic profiles and tissue-specific markers, such as human placental lactogen (hPL). Analysis of expression profiles of five normal ovary and four poorly differentiated serous papillary ovarian adenocarcinoma samples revealed an overall increase in angiogenesis-related markers, including vascular endothelial growth factor (VEGF) and angiopoietin-1 in the diseased tissue. These were accompanied by increases in immune response mediators (e.g. HLA-DR, Ron), apoptotic and neoplastic markers (e.g. BAD protein, b-myb), and novel potential markers of ovarian cancer, such as cofilin, moesin, and neuron-restrictive silencer factor (REST) protein. CONCLUSIONS: In-house production of tailored cDNA arrays, coupled to comprehensive analysis of resulting hybridization profiles, provides an accessible, reliable, and highly effective method of applying array technology to study disease processes. In the ovary, abundance of specific tumor markers, increased macrophage recruitment mediators, a late-stage angiogenesis profile, and the presence of chemoresistance-related markers distinguished normal and advanced ovarian cancer tissue samples. Detection of such parallel changes in pathway- and tissue-specific markers may prove a hallmark ready for application in reproductive disease diagnostic and therapeutic developments.

Biomarkers, Tumor↗

PubMed2000

Angiogenesis and implantation.

Mammalian implantation is a complex, but still poorly understood, process in which the developing embryo establishes contact with the endometrial surface epithelium before developing a haemochorial placenta. The growth and development of the placenta and its subsequent invasion into the decidua can be likened to the invasion of healthy tissue by malignancy. The establishment of an angiogenic phenotype is a critical event in the progression of benign disease to malignant. Little is known about the role of angiogenesis in implantation but in view of the importance of vascular development to the receptive endometrium, angiogenesis is likely to be critical for successful implantation. Here, the factors that control the development of blood vessels in the pre-implantation phase are reviewed and the complex interplay between the embryo and decidua that leads to the establishment of a healthy placenta is considered.

Adaptation, Physiological↗

PubMed1999

Hepatocyte growth factor (HGF) in ovarian epithelial tumour fluids stimulates the migration of ovarian carcinoma cells.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor implicated in the growth and spread of some epithelial tumours. The epithelial cells of a proportion of ovarian tumours, and some ovarian carcinoma cell lines, express high levels of the HGF receptor, c-Met. In this study, we show that ovarian ascitic fluid as well as benign and malignant ovarian cyst fluids contain significant levels of HGF. Ovarian cyst and ascitic fluid stimulated the migration of the ovarian carcinoma cell line, SK-OV-3, and this was greatly reduced by the addition of an HGF-neutralising antibody. Non-malignant peritoneal fluid contained low levels of HGF, and did not stimulate migration of the SK-OV-3 cells. Our results show that HGF is present in benign and malignant ovarian cyst and ascitic fluid, and that HGF in ovarian tumour fluid may be a major inducer of ovarian carcinoma cell migration.

Adult↗

PubMed1999

Randomised controlled trial of educational package on management of menorrhagia in primary care: the Anglia menorrhagia education study.

OBJECTIVE: To determine whether an educational package could influence the management of menorrhagia, increase the appropriateness of choice of non-hormonal treatment, and reduce referral rates from primary to secondary care. DESIGN: Randomised controlled trial. SETTING: General practices in East Anglia. SUBJECTS: 100 practices (348 doctors) in primary care were recruited and randomised to intervention (54) and control (46). INTERVENTIONS: An educational package based on principles of "academic detailing" with independent academics was given in small practice based interactive groups with a visual presentation, a printed evidence based summary, a graphic management flow chart, and a follow up meeting at 6 months. OUTCOME MEASURES: All practices recorded consultation details, treatments offered, and outcomes for women with regular heavy menstrual loss (menorrhagia) over 1 year. RESULTS: 1001 consultation data sheets for menorrhagia were returned. There were significantly fewer referrals (20% v 29%; odds ratio 0. 64; 95% confidence interval 0.41 to 0.99) and a significantly higher use of tranexamic acid (odds ratio 2.38; 1.61 to 3.49) in the intervention group but no overall difference in norethisterone treatment compared with controls. There were more referrals when tranexamic acid was given with norethisterone than when it was given alone. Those practices reporting fewer than 10 cases showed the highest increase in prescribing of tranexamic acid. CONCLUSIONS: The educational package positively influenced referral for menorrhagia and treatment with appropriate non-hormonal drugs.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Cloning and characterization of a beta-galactoside-binding protein (galectin) from the gut of the gastrointestinal nematode parasite Haemonchus contortus.

A cDNA encoding a beta-galactoside-binding lectin (galectin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a membrane protein complex (H-gal-GP) derived from the parasites' gut. The cDNA sequence, exhibiting a tandem repeat structure and designated Hco-gal-2, showed significant levels of similarity with galectins from several species of nematode as well as mammalian galectin type 4. Native galectin was preferentially extracted from the H-gal-GP complex and also from an insoluble membrane fraction prepared from adult worms using lactose-agarose affinity chromatography. The affinity-purified material had apparent molecular mass of around 35 kDa with 3 distinct bands visible on SDS-PAGE. All 3 bands were identified as galectins by reaction with antiserum raised to recombinant Hco-GAL-2 on Western blot. To determine whether H. contortus galectins have any protective capacity against infection lambs were vaccinated with affinity-purified galectin and subsequently given a single challenge infection. Vaccination did not confer any protection against infection with H. contortus as judged by faecal egg output or worm counts.

Amino Acid Sequence↗

PubMed1999

Further immunization and biochemical studies with a protective antigen complex from the microvillar membrane of the intestine of Haemonchus contortus.

Immunization of sheep with a protective antigen complex from the intestinal cells of Haemonchus contortus in Freund's adjuvant stimulated individually variable antibody responses but still conferred significant protection against parasite infection. Correlation between antibody concentration and degree of protection was suggestive of antibody being the effector mechanism. The antigen is known as Haemonchus galactose-containing glycoprotein complex (H-gal-GP) because it binds to lectins with a specificity for N-acetyl-galactosamine. Polypeptide composition analysis by polyacrylamide gel electrophoresis indicated an apparent molecular weight of about 1000 kDa and SDS gels revealed four major polypeptides, containing between 2 and 5 disulphide linked subunits, nearly all being glycosylated. N-terminal amino acid sequence was obtained from 12 subunits, ten showing homologies with cDNAs from Haemonchus encoding either pepsin, metalloprotease or cysteine protease-like enzymes. pH optima, inhibitor and various substrate studies confirmed that the native complex possessed proteolytic activities in agreement with the sequence data. Although the cDNAs predicted water soluble enzymes, little of the complex was solubilized from worm membranes without the use of a detergent, such as Triton X-100. It is hypothesized that H-gal-GP is a gut membrane associated multiprotease complex which is involved in the digestion of the blood meal and which can be neutralized by specific antibodies with drastic consequences for the parasite.

Amino Acid Sequence↗

PubMed1999

Immunization with an affinity purified protein extract from the adult parasite protects lambs against infection with Haemonchus contortus.

Young lambs were immunized with membrane protein extracts from adult Haemonchus contortus which had been enriched for cysteine protease activity using Thiol-Sepharose chromatography. The resulting extract comprised several peptides including cysteine as well as serine and metallo-protease activities although the former predominated. A limited number of the peptide components were glycosylated. Following a single challenge infection with the same parasite, vaccinated lambs had substantially reduced faecal egg outputs and final worm burdens (overall means of 77% and 47%, respectively, from three trials) compared with challenge controls. Antibody in sera from vaccinated lambs was almost exclusively bound to the surface of the parasite gut indicating that protection may be mediated by the inhibition of parasite digestion.

Animals↗

PubMed1999

Two different mutations in the same codon of a type II hair keratin (hHb6) in patients with monilethrix.

Monilethrix is an autosomal dominant hair disorder characterized by a beaded appearance of the hair due to periodic thinning of the shaft. The phenotype shows variable penetrance and results in hair fragility and patchy dystrophic alopecia. Mutations of the helix-encoding region in two hair-specific keratins (hHb1 and hHb6) have been identified. We have now investigated two unrelated monilethrix patients and identified two different novel heterozygous point mutations of the same codon in exon 7 of the hHb6 gene. Dystrophic hair samples obtained from both patients showed the typical beaded appearance by scanning electron microscopy. Both mutations affected the first base of codon 402 (glutamic acid). In patient A, a G to C transition occurred causing a glutamine substitution (GAG to CAG: E402Q) whereas in patient B, the transition was G to A yielding a lysine substitution (GAG to AAG: E402K). The sequence of the 1A helical regions of hHb1 and hHb6 as well as the 2B helical region of hHb1, were normal. Unaffected relatives did not have the hHb6 mutation and this codon was found to be highly conserved showing no alteration in the normal population (100 alleles examined). Both mutations disrupted a Taq I restriction site and restriction fragment length polymorphism analysis showed that a diagnostic 361 bp fragment could confirm the mutation. Thus, two new point mutations of the hair-specific keratin gene hHb6 have been identified in this genetic disease.

Alopecia↗

PubMed1999

The effect of etonogestrel on VEGF, oestrogen and progesterone receptor immunoreactivity and endothelial cell number in human endometrium.

Contraceptive use often leads to disrupted endometrial bleeding patterns in women. In this study, two different contraceptive regimes (Mircette, a monophasic oral contraceptive and Implanon, a long-acting gestagen) were used and their effects on the immunoreactivity of vascular endothelial growth factor (VEGF), oestrogen receptor (ER), progesterone receptor (PR) and endothelial cell number were determined. During the untreated normal cycle, there was a significant increase (P = 0.005) in glandular VEGF immunoreactivity and a significant decrease (P < 0.05) in PR immunoreactivity in the mid- and late secretory phases compared with the proliferative phase. There was a significant positive correlation (gamma = 0.38, P = 0.046) between stromal VEGF immunoreactivity and endothelial cell number. This correlation was also apparent during treatment with Implanon, but not with Mircette. Disrupted bleeding patterns were associated with Implanon and, to a lesser extent, with Mircette. Both contraceptives significantly reduced glandular VEGF immunoreactivity. Implanon significantly increased (P = 0.016) glandular PR staining, but Mircette significantly reduced (P = 0.027) stromal PR staining when compared with secretory before-treatment biopsies. There were no changes in endothelial cell number or glandular or stromal ER during the normal cycle, or with use of either contraceptive. There was no association between the parameters measured with bleeding patterns and histological category.

Adolescent↗

PubMed1999

Localization of leukemia inhibitory factor and its receptor in human placenta throughout pregnancy.

Mice in which the gene that encodes the receptor (R) for leukemia inhibitory factor (LIF) has been deleted show abnormal growth and development of the placenta. This indicates that LIF plays an important role in placental development. The expression of LIF-R and LIF was examined in human trophoblast and decidua using in situ hybridization and immunocytochemistry. LIF-R mRNA and immunoreactivity was localized in villous and extravillous trophoblast throughout pregnancy, and in endothelial cells of the fetal villi. Strong expression of mRNA encoding LIF was detected in decidual leukocytes, which are abundant at the implantation site. Extravillous trophoblast, which invades the maternal decidua, therefore expresses LIF-R as it moves past decidual leukocytes, which express LIF mRNA. The effect of LIF on cultured human trophoblast was examined in vitro. Recombinant human LIF had no effect on [3H]thymidine incorporation by purified extravillous trophoblast, nor on expression of integrins alpha1, alpha5, or beta1 by isolated trophoblast. These results identify fetal endothelial cells and all cells of the trophoblast lineage as targets for the action of LIF in human placenta. Although its effects on trophoblast are not yet clear, LIF appears to mediate interactions between maternal decidual leukocytes and invading trophoblast. LIF may also play a critical role in controlling angiogenesis in the placental villi, since human fetal endothelial cells express LIF-R, and mice lacking a functional LIF receptor gene show altered vascular development in the placenta.

Cell Division↗

PubMed1999

Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity.

Angiogenesis is essential for normal mammalian development and is controlled by the local balance of pro- and antiangiogenic factors. Here we describe a novel mouse cDNA sequence encoding sFLT-1 that is a potent antagonist to vascular endothelial growth factor (VEGF) and show for the first time its in vivo production. In situ hybridization and Northern blot analysis with probes specific for sFLT-1 or FLT-1 showed that the relative abundance of their mRNAs changed markedly in spongiotrophoblast cells in the placenta as gestation progressed. On day 11 of pregnancy, sFLT-1 mRNA was undetectable but FLT-1 readily apparent, and by day 17 sFLT-1 mRNA was abundant but FLT-1 barely detectable. sFLT-1 was identified in conditioned medium of cultured placenta from day 17 pregnant mice and likely to be present in the circulation, as there is a substantial increase of VEGF-binding activity in the serum from day 13 of pregnancy, which coincides with the abundant sFLT-1 expression in placenta. Expression of sFLT-1 was also observed in adult lung, kidney, liver, and uterus. These data suggest a novel mechanism of regulation of angiogenesis by alternative splicing of FLT-1 pre-mRNA. Treatment of pregnant mice with exogenous VEGF from day 9 to 17 of pregnancy, which alters the ratio of VEGF to sFLT-1, resulted in an increase in the number of resorption sites and fibrin deposition in the placenta of ongoing pregnancies. These findings have important implications for understanding placental function and may be relevant in a range of disease states.

Alternative Splicing↗

PubMed1999

Administration of gonadotropins stimulates proliferation of normal mouse ovarian surface epithelium.

Little is known concerning the proliferation of the ovarian surface epithelium or the factors which control this process. To define when and under what circumstances this epithelium proliferates, we have studied the proliferation of mouse ovarian surface epithelium (OSE) during embryogenesis, early postnatal life, various physiological circumstances in the adult and in response to gonadotropic hormones, using the bromodeoxyuridine technique. Proliferation of the OSE is greatest during embryonic development, and falls gradually after birth until sexual maturity is reached. Very little proliferation of the OSE is detectable in adult life in non-pregnant, pregnant or lactating mice. The basal proliferation of the OSE can be increased significantly by inducing follicular development with pregnant mare serum gonadotropin (PMSG) or by administration of the pure recombinant gonadotropins follicle-stimulating hormone (FSH) or luteinizing hormone (LH). These results show that administration of gonadotropins to sexually mature mice induces proliferation of ovarian surface epithelium concurrently with the process of folliculogenesis.

Animals↗

PubMed1998

Activation of large-conductance potassium channels in pregnant human myometrium by pinacidil.

OBJECTIVE: The aim was to investigate the effects of the potassium-channel opener pinacidil on single uterine potassium channels and the contribution of the latter to pinacidil-induced myometrial relaxation. STUDY DESIGN: Myometrial strips and freshly dispersed uterine myocytes were prepared from the myometrial biopsy samples of women undergoing elective, nonlabor caesarean section at term gestation. RESULTS: In isometric tension experiments pinacidil potently relaxed pregnant nonlabor human myometrial strips, with an agonist concentration yielding the half maximal response of 0.4 +/- 0.1 micromol/L. This effect was antagonized by 500 nmol/L charybdotoxin. Application of 10 micromol/L glibenclamide also inhibited the pinacidil-induced relaxation. Coapplication of charybdotoxin (500 nmol/L) and glibenclamide (10 micromol/L) produced a biphasic curve, which was fitted to a two-site model with values for agonist concentration yielding the half maximal response of 0.6 +/- 0.2 micromol/L and 189.7 +/- 0.8 micromol/L. Large-conductance calcium-dependent potassium channel activity was dramatically increased after application of pinacidil (between 10 and 100 micromol/L) to both inside-out and outside-out patches. The activation required the presence of calcium ions at the intracellular aspect of the membrane. Charybdotoxin but not glibenclamide blocked pinacidil-induced unitary large-conductance calcium-dependent potassium channel activity. CONCLUSION: Pinacidil-mediated relaxation of human pregnant myometrial strips may be partially attributable to the opening of uterine large-conductance calcium-dependent potassium channels in addition to adenosine triphosphate potassium channel activation. Drugs with specific potassium channel-activating properties may have important clinical application as novel tocolytics in the treatment of preterm labor.

Calcium↗

PubMed1998

Hepatocyte growth factor levels during normal and intra-uterine growth-restricted pregnancies.

Hepatocyte growth factor (HGF), also known as scatter factor, binds the c-met receptor. It has been shown to be involved in mesenchyme-epithelial interactions. HGF is produced by the villous mesenchyme of the placenta throughout pregnancy and its receptor located on the villous cytotrophoblast cells. In this study the levels of HGF were measured in consecutive samples of plasma taken from pregnant women. Normal pregnancies were compared with intrauterine growth restricted (IUGR) pregnancies (below the third centile). In both groups, the levels of HGF were found to increase significantly as pregnancy progressed and then fall post partum. There was a considerable amount of variation found between individual women but no significant difference (P=0.65) between the normal and IUGR pregnancies.

Adult↗

PubMed1998

Contribution of calcium-sensitive potassium channels to NS1619-induced relaxation in human pregnant myometrium.

Large-conductance, calcium-sensitive potassium channels (BK(Ca)) are found at high density in the pregnant human myometrium. We have investigated, using isolated myometrial strips and freshly dispersed human myometrial cells, the action of a novel drug (NS1619) which has potassium channel opening activity. Isometric tension experiments demonstrated that NS1619 has a potent, relaxant effect on the pregnant human myometrium. Using both the inside-out and outside-out configurations of the patch-clamp technique, NS1619 appears to act directly on myometrial BK(Ca) channels to stimulate channel activity by increasing the time spent in the open state by this group of potassium channels. Consequently, the myometrial BK(Ca) channel may be a novel target site for drug intervention in clinical conditions, e.g. failure to labour, preterm labour or dysmenorrhoea, which may require either the augmentation or inhibition of uterine K+ channel activity.

Benzimidazoles↗