Investigation of factors influencing the immunogenicity of hCG as a potential cancer vaccine.

Human chorionic gonadotrophin (hCG) and its ß-subunit (hCGß) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C-terminal hCGß peptide (hCGßCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGß mutant (hCGßR68E, designed to eliminate cross-reactivity with luteinizing hormone) or hCGßCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross-linkers [1-ethyl-3(3-dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGßR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGßCTP peptide irrespective of the carrier used, how it was cross-linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self-protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGßR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGßCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGß-positive cancer patients.

Limited evolutionary conservation of imprinting in the human placenta.

The epigenetic phenomenon of genomic imprinting provides an additional level of gene regulation that is confined to a limited number of genes, frequently, but not exclusively, important for embryonic development. The evolution and maintenance of imprinting has been linked to the balance between the allocation of maternal resources to the developing fetus and the mother's well being. Genes that are imprinted in both the embryo and extraembryonic tissues show extensive conservation between a mouse and a human. Here we examine the human orthologues of mouse genes imprinted only in the placenta, assaying allele-specific expression and epigenetic modifications. The genes from the KCNQ1 domain and the isolated human orthologues of the imprinted genes Gatm and Dcn all are expressed biallelically in the human, from first-trimester trophoblast through to term. This lack of imprinting is independent of promoter CpG methylation and correlates with the absence of the allelic histone modifications dimethylation of lysine-9 residue of H3 (H3K9me2) and trimethylation of lysine-27 residue of H3 (H3K27me3). These specific histone modifications are thought to contribute toward regulation of imprinting in the mouse. Genes from the IGF2R domain show polymorphic concordant expression in the placenta, with imprinting demonstrated in only a minority of samples. Together these findings have important implications for understanding the evolution of mammalian genomic imprinting. Because most human pregnancies are singletons, this absence of competition might explain the comparatively relaxed need in the human for placental-specific imprinting.

Imprinting of IGF2 P0 transcript and novel alternatively spliced INS-IGF2 isoforms show differences between mouse and human.

Genomic imprinting is limited to a subset of genes that play critical roles in fetal growth, development and behaviour. One of the most studied imprinted genes encodes insulin-like growth factor 2, and aberrant imprinting and DNA methylation of this gene is associated with the growth disorders Beckwith-Wiedemann and Silver-Russell syndromes and many human cancers. Specific isoforms of this gene have been shown to be essential for normal placental function, as mice carrying paternal null alleles for the Igf2-P0 transcript are growth restricted at birth. We report here the identification of three novel human transcripts from the IGF2 locus. One is equivalent to the mouse Igf2-P0 transcript, whereas the two others (INSIGF long and short) originate from the upstream INS gene that alternatively splices to downstream IGF2 exons. In order to elucidate the molecular mechanisms involved in the complex imprinting of these novel IGF2 transcripts, both the allele-specific expression and methylation for all the IGF2 promoters including P0 and the INSIGF transcripts were analysed in human tissues. Similar to the mouse, the human IGF2-P0 transcript is paternally expressed; however, its expression is not limited to placenta. This expression correlates with tissue-specific promoter methylation on the maternal allele. The two novel INSIGF transcripts reported here use the INS promoter and show highly restricted tissue expression profiles including the pancreas. As previously reported for INS in the yolk sac, we demonstrate complex, tissue-specific imprinting of these transcripts. The finding of additional transcripts within this locus will have important implications for IGF2 regulation in both cancer and metabolism.

Regulation of human villous trophoblast by insulin-like growth factors and insulin-like growth factor-binding protein-1.

Many studies have implicated the insulin-like growth factors (IGFs) and insulin-like growth factor-binding protein-1 (IGFBP-1) in the control of the feto-maternal interface of human pregnancy, but many of the data are from cell lines derived from primary trophoblast or from extravillous trophoblast. We have obtained highly enriched villous cytotrophoblast (VCT) from first trimester and term human placentae, and investigated the effects of IGF-I, IGF-II and phosphoisoforms of IGFBP-1. First trimester villous trophoblast cells were regulated by all these factors. IGF-II increased cell numbers 3.5-fold after 96 h in culture, and IGF-I had less effect (1.5-fold increase) (both P<0.05). IGF-II also had a greater effect on the levels of matrix metalloproteinase (MMP)-2 and MMP-9. Phosphorylated and non-phosphorylated iso-forms of IGFBP-1 added alone increased cell numbers and MMP levels (P<0.05). IGFBP-1 did not modify the effects of IGF-II on cell numbers or on MMP production. Term VCT numbers and MMP production in vitro were unaffected by IGFs (P>0.05). Cell numbers were increased only by 100 nM IGFBP-1 isoforms (P<0.05), whereas MMP levels released from term cells were optimally increased by 1-10 nM IGFBP-1. Overall, our data show that IGFs regulate only first trimester, but not term, VCT. IGFBP-1 regulates VCT from both gestations, but the effects are concentration and end-point specific. In particular, first trimester cell numbers are more affected by low levels of IGFBP-1, whereas high levels of IGFBP-1 are needed to increase MMP and the converse applies to term VCT; low levels of IGFBP-1 have more effect on MMP levels.

The relationship between insulin and insulin-like growth factor binding protein-1 is modified by pre-eclampsia.

Insulin is the main negative regulator of insulin-like growth factor binding protein-1 (IGFBP-1) in the non-pregnant state. Although changes in insulin resistance and circulating level of IGFBP-1 occur in pre-eclampsia, little is known about the relationship between insulin and IGFBP-1 in pregnancies complicated by the disease. In this study, we have investigated whether the relationship between insulin and IGFBP-1 is modified by pre-eclampsia. Maternal levels of insulin and IGFBP-1 were measured, at 4-weekly intervals between 16 and 36 weeks' gestation, in plasma samples obtained from ten normal pregnant controls and ten women who developed pre-eclampsia. The controls were chosen to be similar in maternal age and booking body mass index to the pre-eclampsia group. Insulin levels increased in both the normal controls and the women who developed pre-eclampsia. The levels in pre-eclampsia were significantly greater than those in normal pregnancy at 32 and 36 weeks' gestation (p = 0.02 and 0.005, respectively). IGFBP-1 levels were unchanged in normal pregnancy and rose in pre-eclampsia. In normal pregnancy, insulin levels were inversely related to IGFBP-1 levels throughout. In women developing pre-eclampsia, the relationship between insulin and IGFBP-1 was negative at 16 weeks and positive from 24 weeks. These data suggest that whereas the inverse relationship between insulin and IGFBP-1 is maintained during normal pregnancy, this relationship is reversed in women who develop pre-eclampsia.

Regulated expression of signal transducer and activator of transcription, Stat5, and its enhancement of PRL expression in human endometrial stromal cells in vitro.

Differentiation of human endometrium during the secretory phase of the menstrual cycle is characterized by expression of a variety of genes implicated in the establishment and maintenance of pregnancy. An increased abundance of signal transducers and activators of transcription (Stats) in the secretory phase suggests Stat5 as a component of the differentiation of endometrium in response to ovarian hormone stimulation in vivo. Decidualization is initiated in a subset of endometrial stromal cells (ESC) in vivo during the secretory phase, but it is unclear whether regulated expression of Stat5 is a feature of these cells. Here, therefore, the abundance and subcellular distribution of Stat5 in ESC after a decidualization stimulus of cAMP plus medroxyprogesterone acetate (MPA) has been investigated in vitro. Western blotting revealed an increase in the apparent abundance of Stat5a and Stat5b, in the cytosolic and nuclear fractions, at 2, 3, and 4 d after stimulation. The potential functional relevance of this increase in Stat5 is suggested by the ability of transiently transfected Stat5a or Stat5b to significantly enhance the response of the decidual PRL promoter to cAMP/MPA and attenuation of the response to cAMP/MPA by dominant negative Stat5. Recent evidence suggests endometrial differentiation, including PRL production, as a possible target of antiphospholipid antibodies (aPL) prevalent in recurrent miscarriage. Monoclonal antibody, ID2, which has similar reactivity as human aPL, significantly decreased the apparent abundance of nuclear Stat5b in response to cAMP/MPA and was associated with decreased decidual PRL promoter activation and PRL secretion. Regulated expression of Stat5 is therefore a component of decidual differentiation of human ESC and contributes significantly to activation of the decidual PRL promoter. Alteration of this process by an aPL component suggests decidual differentiation as a potential clinical target in recurrent early miscarriages.

A longitudinal study of maternal plasma insulin-like growth factor binding protein-1 concentrations during normal pregnancy and pregnancies complicated by pre-eclampsia.

Insulin-like growth factor binding protein-1 (IGFBP-1) is synthesized by the decidual stroma, and is thought to act locally to inhibit IGF activity and so limit trophoblast invasion. Cross-sectional studies have reported conflicting data on maternal circulating concentrations of IGFBP-1 in early pregnancy before the development of pre-eclampsia. A longitudinal study was performed in 10 women who went on to develop pre-eclampsia and a group of 12 normal pregnant controls, chosen to be similar for maternal age, booking body mass index (BMI) and gestational age. Maternal IGFBP-1 concentrations were measured in plasma obtained at 16, 20, 24, 28, 32 and 36 weeks. Plasma IGFBP-1 concentrations were unchanged over this period in normal pregnancy. In contrast, the concentrations in women who developed pre-eclampsia increased progressively. At 16, 20, and 24 weeks the concentrations were significantly lower compared to normal pregnancy, at 28 and 32 weeks, similar, but by 36 weeks the concentrations were significantly greater than the normal controls. The data show that circulating IGFBP-1 concentrations are lower in early pregnancy before the development of pre-eclampsia. Thus, it is suggested that IGFBP-1-induced inhibition of IGF activity is unlikely to be responsible for the impaired trophoblast invasion observed in pre-eclampsia.

Antenatal dexamethasone therapy does not affect circulating concentrations of insulin-like growth factor binding protein-1.

In animals, dexamethasone administration during pregnancy leads to fetal growth restriction due to enhanced expression of insulin-like growth factor binding protein-1 (IGFBP-1). In humans, there is also a significant inverse correlation between maternal and fetal concentrations of IGFBP-1 and birth weight. During pregnancy, maternal IGFBP-1 is derived from the decidualized endometrium. We have studied the effect of dexamethasone on circulating concentrations of IGFBP-1 in 12 pregnant women who received dexamethasone therapy for fetal lung maturation in anticipation of premature delivery before 34 completed weeks of gestation. Blood samples were collected before dexamethasone administration, at 24 h and 48 h after the course of dexamethasone, and within 24 h of delivery, for the measurement of IGFBP-1. There was no significant change in plasma IGFBP-1 concentrations at 24 and 48 h following dexamethasone therapy, and at delivery (P = 0.666, 0.307 and 0.398, respectively). Therefore, antenatal dexamethasone therapy does not influence decidual synthesis of IGFBP-1.

Mechanism of action of the intrauterine contraceptive device: evidence for a specific biochemical deficiency in the endometrium.

The precise mechanism of action of the intrauterine contraceptive device (IUCD) is uncertain. In this study we compared the circulating concentrations of a specific endometrial protein, placental protein 14 (PP14), in 62 women with an IUCD and 16 controls. The concentrations of PP14 were substantially lower in IUCD users. There was no difference in the concentrations of another and less specific endometrial protein, insulin-like growth factor binding protein-1 (IGFBP-1). There was no difference in PP14 concentrations between those women with and without intermenstrual bleeding. We conclude that the reduced concentrations of PP14 in IUCD users reflect defective endometrial function in these women, probably related to the contraceptive effect. We propose that the measurement of PP14 might be a means of comparing the efficiency of different devices.

Circulating levels of IGF-I and IGF-binding protein-1 throughout pregnancy: relation to birthweight and maternal weight.

Serum levels of IGF-I and IGF-binding protein (IGFBP-1) have been determined in the maternal circulation between 11 and 42 weeks of gestation in women not in labour (n = 335) and in the maternal and fetal circulations at the time of delivery between 37 and 42 weeks (n = 55). Maternal serum (MS) IGF-I levels increased during pregnancy and showed a significant positive correlation with maternal weight (P = 0.0033) but no correlation with birthweight. The MS IGFBP-1 levels did not change during the second and third trimesters and showed a negative correlation with birthweight, maternal weight, placental weight and MS glucose (P = 0.0002, P < 0.0001, P = 0.047, P = 0.024 respectively). MS IGFBP-1 levels were higher in small-for-gestational age babies than in average-for-gestational weight babies (P = 0.026) and lower in the large-for-gestational weight group (P = 0.048). There was a significant rise in mean MS IGFBP-1 levels during labour (P = 0.0005). These findings suggest that IGFBP-1 may be an important factor in pathological growth retardation.

IGFBP-1 in the placenta, membranes and fetal circulation: levels at term and preterm delivery.

Samples of maternal blood, amniotic fluid, placenta, fetal membranes and umbilical venous blood were collected from 59 women at vaginal delivery (32-41 weeks gestation) and 15 women at delivery by Caesarean section (37-41 weeks gestation). Umbilical vein levels of IGFBP-1 were significantly lower in deliveries prior to the onset of labour (elective Caesarean section) than those during normal vaginal delivery. These levels were, in turn, significantly lower than those delivered by emergency Caesarean section. This difference was not seen in any of the other tissues examined. Concentrations of IGFBP-1 were lower in placenta and membrane extracts from preterm deliveries than in term deliveries. This difference was not observed in maternal or fetal serum or in amniotic fluid. This study confirms that the fetal membranes are a major source of IGFBP-1 and that fetal circulating levels are raised where there is evidence of fetal hypoxia. The absence of a comparable rise in levels in placenta, membranes or amniotic fluid suggests that the origin of this increase is from fetal tissue.

Raised circulating levels of interleukin-6 in women with an intrauterine contraceptive device.

The aim of this study was to determine whether the presence of an intrauterine contraceptive device (IUCD) causes a systemic elevation of interleukin-6 (IL-6). The control group comprised 29 women with normal cycles and who were not using any form of contraception and the study group comprised 74 women who had had an IUCD in place for 10-24 months. Of these, 45 had intermenstrual bleeding. Systemic IL-6 was undetectable (minimum detection limit of the assay is 5 pg/ml) in the control but was raised in 20% of the women fitted with an IUCD (p < 0.01). There was no difference between IL-6 levels in IUCD users who had intermenstrual bleeding and those who did not. These results suggest that the effects of chronic IUCD usage may not be limited to the endometrium.

PIP: A case-control study indicated that IUD use causes a systemic elevation of interleukin-6 (IL-6) comparable to that observed in acute and chronic inflammatory conditions. The study group was comprised of 74 women who had had a Copper T-380A device in place for 10-24 months; 45 of these women reported IUD-related intermenstrual bleeding. 29 women with normal cycles who were not using any form of contraception served as controls. IL-6 levels were assessed through immunoradiometric assay. All 29 controls had undetectable circulating IL-6 levels, while 15 (20%) of cases had raised systemic levels of this cytokine. There were no differences in circulating IL-6 levels between cases who did and did not experience intermenstrual bleeding, nor was there a correlation between IL-6 levels and the day of the cycle on which a blood sample was randomly collected. It is unclear whether the circulating IL-6 observed in this study is an "overspill" from locally produced IL-6 or indicative of a more generalized systemic immunoactivation. Recommended are studies that monitor local and systemic production of IL-6 in IUD users in conjunction with other parameters of an inflammatory response, including C-reactive protein.

Elevated levels of insulin-like growth factor binding protein-1 in fetal distress.

OBJECTIVE: To investigate the association between fetal distress (abnormal cardiotocograph tracing and/or a low fetal pH) and the levels of fetal IGFBP-1. DESIGN: Prospective comparative study. SUBJECTS AND METHODS: Twenty-two women in labour with evidence of fetal distress defined by FIGO criteria and 19 women in uncomplicated labour. The gestation range was 37 to 42 weeks and birthweight range was 2500 to 4240 g. IGFBP-1 was determined by radioimmunoassay. RESULTS: The umbilical levels of IGFBP-1 were significantly higher in the study group compared with the control group (median 282.5 micrograms/l versus 128 micrograms/l, P = 0.0046; Mann-Whitney U test). There was a significant inverse correlation between fetal IGFBP-1 and cord pH (r = 0.58, P < or = 0.0001). There was no difference between the maternal serum levels of IGFBP-1 in the two groups. CONCLUSION: Umbilical IGFBP-1 is elevated in association with fetal distress.

The effects of labor on maternal and fetal levels of insulin-like growth factor binding protein-1.

OBJECTIVE: Our purpose was to determine the effects of labor and fetal hypoxia on the levels of insulin-like growth factor binding protein-1 in the maternal and fetal circulation. STUDY DESIGN: Serum levels of insulin-like growth factor binding protein-1 were determined in maternal and umbilical blood at delivery in two groups. The first group included 43 vaginal deliveries and 23 elective cesarean sections. The second group consisted of 44 women; in 24 the liquor was meconium stained and in 20 it was clear. RESULTS: Levels of insulin-like growth factor binding protein-1 in the neonate were lower in deliveries occurring before onset of labor (p < 0.001), Mann-Whitney U test) and higher in cases with severe meconium staining (p = 0.01). There were no differences in maternal levels of insulin-like growth factor binding protein-1 between subjects in labor and not in labor or those with or without meconium staining. CONCLUSION: The process of labor leads to an increase in fetal levels of insulin-like growth factor binding protein-1. This increase may well be associated with the relative fetal stress that occurs during labor. This suggestion is supported by the finding of the highest levels in labors in which there was thick staining of the liquor.

Strontium isotopes in deep-sea sediments.

Recent deep-sea sediments, leached of carbonate, have Sr(87) Sr(86) ratios ranging from 0.7044 to 0.7394. Strontium in the detrital sediment has not equilibrated isotopically with seawater strontium. Amounts of strontium-bearing authigenic material in the clay-mineral assemblage are not great enough to mask strontium-isotope ratios inherited from source areas.