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Biomedical subjects

D S Charnock-Jones

Publications and source records attributed to D S Charnock-Jones.

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

The role of leukemia inhibitory factor and interleukin-6 in human reproduction.

There is now strong evidence that many of the actions of steroids in controlling reproduction are mediated by locally acting factors such as growth factors and cytokines. These have been shown to act both in an autocrine and paracrine manner to regulate preimplantation embryo development and migration which is necessary for placental development. The creation of mouse strains lacking genes for receptors or growth factors has proved important in defining which of these are essential in reproduction in this species and those that play a lesser role. Using this approach, a lack of leukemia inhibitory factor (LIF) in the murine endometrium has been shown to result in failed implantation. Evidence from infertile women supports the notion that abnormal expression of LIF, or the related cytokine interleukin-6 (IL-6) in the endometrium may underlie some forms of human infertility. This offers the opportunity for therapeutic intervention, if levels of these cytokines can be altered in a specific and controlled way. The recently described method of delivery of genes to the uterine epithelium provides a powerful new approach by which this could be achieved. The ability to regulate the function of specific genes in the endometrium by direct gene transfer raises the prospect of novel opportunities for therapeutic intervention.

Animals↗
PubMed1998

A vascular endothelial growth factor antagonist is produced by the human placenta and released into the maternal circulation.

Vascular endothelial growth factor (VEGF) is a potent secreted factor that promotes angiogenesis and maintains the integrity of the endothelium. Levels of VEGF are increased in many tumors and are elevated in women with pre-eclampsia, a serious disease of pregnancy. Here we show by in situ hybridization that the trophoblast contains the mRNA encoding a soluble version of the VEGF receptor known as Flt-1 (sFlt-1: initially described by Kendall and Thomas, PNAS 90:10705-10709). Binding assays and Western blotting of villus-conditioned media confirmed the production of sFlt-1. Serum from pregnant women was found to contain a VEGF-binding protein that was not present in serum from men or nonpregnant women. As determined by heparin affinity, column fractionation, and cross-linking, this protein was identical to sFlt-1. Taken together, these results show that the placenta secretes sFlt-1, which would be expected to be a VEGF antagonist. This is the first report of production of the sFlt-1 receptor in vivo, and it reveals a new mechanism for naturally regulating this potent angiogenic agent. The presence of such an antagonist suggests that regulation of VEGF action is essential to successful pregnancy. This has important implications for the activity of VEGF locally and systemically in other conditions.

Blotting, Western↗
PubMed1998

Expression and localization of the Th2-type cytokine interleukin-13 and its receptor in the placenta during human pregnancy.

PROBLEM: To investigate the expression of the Th2-type cytokine interleukin (IL)-13 and its receptor in human placenta during gestation. METHOD OF STUDY: Expression of IL-13 and its receptor was analyzed by reverse transcriptase (RT)-polymerase chain reaction (PCR), in situ hybridization and immunohistochemistry using human placental samples. RESULTS: IL-13 mRNA was detected by RT-PCR in placental extracts from all stages of gestation. In situ hybridization revealed IL-13 mRNA in first trimester cytotrophoblast and syncytiotrophoblast. Few positive cells were found within decidual sections from the same pregnancy. Immunohistochemistry revealed a similar pattern to in situ hybridization. This was largely absent in second- and third-trimester placentae. IL-13 receptor alpha chain (IL-13R alpha) was detected by immunofluorescence on the surface of leukocytes in first-trimester villous core and decidua. CONCLUSIONS: The expression of IL-13 is spatially and temporally regulated in the placenta, and associated with the close proximity of a receptor-bearing target leukocyte population.

DNA, Complementary↗
PubMed1998

Comparison of expression patterns for placenta growth factor, vascular endothelial growth factor (VEGF), VEGF-B and VEGF-C in the human placenta throughout gestation.

Angiogenesis and vascular transformation are important processes in the normal development of the placenta. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor and is thought to be important for placental development. Recently several new members of this family have been described. In this study we used in situ hybridisation to localise which cells in the placenta expressed mRNA for VEGF, placenta growth factor (PlGF), VEGF-B and VEGF-C. We were unable to find any message for either VEGF-B or VEGF-C in the placenta, suggesting that only low levels are produced which this method was unable to detect. The mRNA encoding VEGF was found to be produced by cells within the villous mesenchyme, decidual macrophages and decidual glands but, in contrast to our previous findings, not by trophoblast. The mRNA encoding PlGF was produced in large amounts by villous cytotrophoblast, syncytiotrophoblast and extravillous trophoblast. The mRNAs encoding VEGF and PlGF were thus not co-localised and it appears that there is unlikely to be any significant production of VEGF/PlGF heterodimer in the placenta.

Blotting, Northern↗
PubMed1997

Immunolocalization of the apoptosis regulating proteins Bcl-2 and Bax in human endometrium and isolated peritoneal fluid macrophages in endometriosis.

Endometriosis, a debilitating disease associated with infertility, is characterized by the prolonged presence of ectopic endometrial tissue and the involvement of activated peritoneal fluid macrophages. Apoptosis, which occurs in both endometrium and peritoneal fluid macrophages, is controlled in part by members of the Bcl-2/Bax family of proteins. Here, through immunohistochemical staining, we investigated the Bcl-2/Bax status in endometrium and peritoneal fluid macrophages in endometriosis. Bcl-2/Bax immunoreactivity was found predominantly in the glandular epithelial cells, mainly during the proliferative phase of the menstrual cycle for Bcl-2 but throughout the entire menstrual cycle for Bax. Ectopic endometrium contained a population of Bcl-2 positive. Bax negative tissue macrophages. Fluorescence-activated cell sorting of isolated peritoneal fluid macrophages showed that women with endometriosis had a significantly higher proportion of Bcl-2 positive macrophages than the non-endometriotic group. The proportion of Bax positive peritoneal fluid macrophages was significantly elevated in women without endometriosis. The increased proportion of Bcl-2 positive macrophages found in women with endometriosis may predispose these cells to resist apoptosis. The continued survival of these active cells could have important consequences for the survival and proliferation of the ectopic endometrial tissue.

Adult↗
PubMed1997

In-vivo gene transfer to the uterine endometrium.

Therapeutic interventions in reproductive biology have relied largely on steroids and antisteroids which act to regulate gene expression in target tissues. Whilst their use has transformed women's lives, few conceptual advances have been made in contraceptive technology, no means identified to improve human implantation and no new strategies developed for the treatment of benign gynaecology. A novel alternative is direct gene transfer to the organ of interest. As a first step to achieving this goal in the uterus, we used reporter gene constructs to transfect mouse endometrium in vivo and human endometrial epithelial cells in vitro. We injected DNA-liposome complexes into the uterine lumen of mice on day 2 of pseudopregnancy and detected reporter gene activity 2 days later. The liposomes used were a 3:1 (w/w) mixture of 2,3-dioleyloxy-N-[2(sperminecarboxamido) ethyl]-N-N-dimethyl-1-propanaminium trifluoroacetate and dioleoylphosphatidyl ethanolamine. Freshly isolated human endometrial epithelial cells were successfully transfected in vitro with similar DNA-liposome complexes. These data suggest that endometrial gene transfer may be effective in humans. This may lead to the development of new therapeutic agents, including contraceptives, for the improvement of women's health.

Animals↗
PubMed1997

Cyclooxygenase-1 and -2 in human placenta and placental bed after normal and pre-eclamptic pregnancies.

In pre-eclampsia, the ratio of prostacyclin:thromboxane production rate is decreased favouring the vasoconstrictive thromboxane. One of the rate-limiting steps in prostaglandin synthesis is cyclooxygenase (COX) activity. Therefore, we investigated the expression of COX-1 and COX-2 in human placenta and placental bed. Tissue specimens from the 29th to 40th week of pregnancy were obtained from Caesarean sections after uncomplicated and pre-eclamptic pregnancies before the onset of labour. COX-1 and COX-2 were localized immunohistochemically with the identification of positive cells by double immunofluorescence staining. The protein and mRNA levels were analysed by immunoblotting and quantitative reverse transcriptase-polymerase chain reaction. Expression of both COX-1 and COX-2 could be observed in placenta and placental bed. COX-1-like immunoreactivity was observed in most cell types with strongest staining in macrophages. Only macrophages, endothelium, vascular leiomyocytes and fibroblasts stained positively for COX-2. In placenta, COX-1 and -2 expression was unchanged after pre-eclampsia. In placental bed, protein and mRNA levels of COX-1 were increased in the pre-eclamptic group (P < 0.05), whereas COX-2 expression did not differ significantly from normal pregnancies. An increased expression of COX-1 could be involved in the pathophysiology of pre-eclamptic changes within the placental bed. A therapy with drugs inhibiting COX-1 might be beneficial in this condition.

Blotting, Western↗
PubMed1997

Distribution of the A and B forms of the progesterone receptor messenger ribonucleic acid and protein in uterine leiomyomata and adjacent myometrium.

The two forms of the progesterone receptor, PR-A and PR-B, are independently regulated at the transcriptional level, and show distinct responses to progesterone antagonists. We were interested in possible differences in the PR-A to PR-B ratio between uterine myometrium and leiomyomata (fibroid), that might influence the response of fibroids to progesterone agonists and antagonists, and thus have consequences for the treatment of this condition. Fibroid and adjacent normal myometrium were obtained from 11 women undergoing hysterectomy. Immunohistochemistry using a monoclonal antibody which recognizes both PR-A and PR-B showed exclusively nuclear staining, and this was stronger in the leiomyomata than in adjacent myometrium. An antibody specific for PR-B gave fainter staining of both tissues. Western blotting confirmed a higher concentration of PR in leiomyomata than myometrium in eight out of 11 cases. In all cases both forms were present, with a consistent dominance of PR-A over PR-B. However an RNase protection assay showed that there was no difference between the concentrations of mRNA encoding PR-A and PR-B, or between the mRNA concentrations in leiomyomata and normal myometrium. We conclude that the observed differences between the levels of immunoreactive PR in leiomyomata and myometrium may result from post-translational control, and support the use of progesterone antagonists in the treatment of leiomyomata.

Adult↗
PubMed1997

Isolation of macrophages (Hofbauer cells) from human term placenta and their prostaglandin E2 and thromboxane production.

Placental macrophages (Hofbauer cells) are located close to trophoblast cells and fetal capillaries, which makes them ideal candidates for involvement in regulatory processes within the villous core. Their production of various cytokines and prostaglandin (PG) synthesizing enzymes has previously been shown immunohistochemically. Hofbauer cells were isolated from human placenta after term deliveries by Ficoll and Percoll gradient centrifugation. Remaining trophoblast cells were removed with anti-epidermal growth factor (EGF)-receptor-coated Dynabeads followed by differential adherence. The identity of isolated cells was investigated by immunohistochemistry with anti-CD68, which showed that >90% cells were positive. After a 36 h recovery period in either 20% O2 or 5% O2, fresh medium was applied and PGE2 and thromboxane (TXA2) production analysed by enzyme immunoassay at 4, 8, and 24 h. PGE2 and TXA2 were both produced by placental macrophages with PGE2 synthesis being predominant. Concentrations of both could be stimulated by lipopolysaccharide with maximum effect after 24 h. Culture in low oxygen caused decreased PGE2 concentrations, whereas TXA2 production remained unchanged. In conclusion, the presented isolation protocol allows further study of Hofbauer cell function. This study also presents novel findings regarding the prostaglandin production of term Hofbauer cells under normal and hypoxic conditions.

Cell Separation↗
PubMed1997

Decreased levels of the potent regulator of monocyte/macrophage activation, interleukin-13, in the peritoneal fluid of patients with endometriosis.

Endometriosis is characterized by an increase in the number, activation and secretory activity of peritoneal fluid macrophages. Factors regulating the activation of these cells may be important in the pathophysiology of this disease. In this study we measured by enzyme-linked immunosorbent assay the concentrations of the macrophage inhibitory factor interleukin (IL)-13 in the peritoneal fluid of women with and without endometriosis. It was found that women with endometriosis had significantly lower amounts of IL-13 (95 +/- 9.8 pg/ml) in peritoneal fluid, compared with women without endometriosis (115 +/- 30 pg/ml) (P < 0.01). No cycle-specific variation was evident for either group. Another macrophage inhibitory interleukin (IL-10) was also measured, but no differences between women with (16.1 +/- 13.2 pg/ml) or without (10.3 +/- 5.6 pg/ml) endometriosis were seen. The immunolocalization of IL-13 was assessed in eutopic and ectopic endometrium and in isolated peritoneal fluid cells. Glandular epithelial cells and stromal cells in both eutopic and ectopic endometrium were immunopositive for IL-13. No cycle-specific differences in the immunolocalization of IL-13 were seen. In conclusion, the reduced amounts of IL-13 in the peritoneal fluid of women with endometriosis may lead to a lack of suppression of macrophage activation, thereby contributing to the overall pathogenesis of this disease.

Adult↗
PubMed1997

Expression and localization of the vascular endothelial growth factor family in ovarian epithelial tumors.

Vascular endothelial growth factor (VEGF) is an angiogenic factor secreted by various tumors, including epithelial tumors of the ovary, and is involved in tumor progression and maintenance. The significance and function of other members of the VEGF family in the ovary has not yet been elucidated. In the present study, we have defined the expression of mRNA encoding VEGF-B, VEGF-C, and placenta growth factor (PIGF), compared with that of VEGF mRNA, in normal ovary and a range of ovarian epithelial tumors. Analysis by reverse transcription-PCR indicated that mRNA encoding VEGF (isoforms 121 and 165), VEGF-B (isoforms 167 and 186), and VEGF-C, but not PIGF, were present in all ovarian tissues examined. By in situ hybridization, neither VEGF-C nor PIGF transcripts were detected in any of the samples. The expression pattern of VEGF-B mRNA was generally similar to that of VEGF mRNA, in that transcripts were readily detected in the epithelial cells of all histologic types of ovarian carcinoma, but could not be detected in normal or benign tumor epithelium. Specific differences in the expression of the two genes were noted in areas of tumor necrosis, in which the expression of VEGF mRNA, but not VEGF-B mRNA, was further enhanced, and in a sample in which VEGF-B mRNA was strongly expressed in tumor-associated macrophages that did not hybridize with the riboprobe to VEGF mRNA. These results imply that a second member of the VEGF family, VEGF-B, may play a significant role in the angiogenesis, progression, and maintenance of ovarian carcinomas.

Adult↗
PubMed1996

Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids.

Angiogenesis is important in the pathophysiology of endometriosis, a condition characterized by implantation of ectopic endometrium in the peritoneal cavity. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in physiological and pathological angiogenesis, and elevated levels of VEGF are found in peritoneal fluid of patients with endometriosis. Our aim was to investigate the site of expression and regulation of VEGF in endometriosis. VEGF immunoreactivity was found in tissue macrophages present in ectopic endometrium and in activated peritoneal fluid macrophages. Macrophage activation was highest in women with endometriosis, and media conditioned by peritoneal fluid macrophages from these women caused a VEGF-dependent increase in endothelial cell proliferation above that seen from normal women. Peritoneal fluid macrophages secreted VEGF in response to ovarian steroids, and this secretion was enhanced after activation with lipopolysaccharide. Peritoneal fluid macrophages expressed receptors for steroid hormones. VEGF receptors flt and KDR (kinase domain receptor) were also detected, suggesting autocrine regulation. During the menstrual cycle, expression of flt was constant but that of KDR was increased in the luteal phase, at which time the cells migrated in response to VEGF. KDR expression and the migratory response were significantly higher in patients with endometriosis. This study demonstrates that activated macrophages are a major source of VEGF in endometriosis and that this expression is regulated directly by ovarian steroids.

Adult↗
PubMed1996

Cloning, expression and genomic organization of human placental protein disulfide isomerase (previously identified as phospholipase C alpha).

Phosphoinositol-specific Phospholipase C plays an important role in transducing receptor generated signals to the rest of the cell. A cDNA encoding a phospholipase has been described (Bennett et al., 1988, Nature 334, 268-270). However it is probable that this cDNA in fact encodes a protein disulfide isomerase. Since the original work suggested that this enzyme was important in the reproductive tract we sort to clone, sequence, express and characterize the recombinant protein isolated from the placenta. We have cloned and sequenced the cDNA encoding the human homolog of this cDNA from human placenta, although the mRNA was widespread in the female reproductive tract. We have transiently expressed it in both COS cells and also 1BR fibroblasts. Cell lysates were assayed for increased phospholipase activity and protein disulfide activity. We describe the entire cDNA sequence which is highly conserved between species. We have also cloned a portion of the genomic gene and described the intron/exon boundaries. In vitro translation of this cDNA showed that it encoded a protein of 61 kD with a cleavable signal peptide. Transient expression showed the protein produced had no phospholipase activity but did show protein disulfide isomerase activity. The expression work shows that this cDNA indeed encodes a protein disulfide isomerase and not a phospholipase. The nucleotide sequence shows marked conservation of the coding and regulatory regions which may suggest that this enzyme has evolved to perform a highly specialized function.

Base Sequence↗