Biomedical subjects

Judith N Bulmer

Publications and source records attributed to Judith N Bulmer.

At least 19 recordsLinked to original sources

PubMed2006

Effects of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on cytokine production by human decidual cells.

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) is a potent immunomodulatory seco-steroid. We have demonstrated that several components of vitamin D metabolism and signaling are strongly expressed in human uterine decidua from first trimester pregnancies, suggesting that locally produced 1,25(OH)(2)D(3) may exert immunosuppressive effects during early stages of gestation. To investigate this further, we used primary cultures of human decidual cells from first and third trimester pregnancies to demonstrate expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)(2)D(3), 1alpha-hydroxylase (CYP27B1). Synthesis of 1,25(OH)(2)D(3) was higher in first trimester decidual cells (41 +/- 11.8 fmoles/h/mg protein) than in third trimester cells (8 +/- 4.4 fmoles/h/mg protein; P < 0.05). Purification of decidual cells followed by quantitative RT-PCR analysis showed that CYP27B1 was expressed by both CD10(+VE) stromal-enriched and CD10(-VE) stromal-depleted cells, with higher levels of mRNA in first trimester pregnancies. Expression of CYP27B1 correlated with TLR4 and IDO. Functional responses to 1,25(OH)(2)D(3) were studied using CD56(+VE) natural killer (NK) cells isolated from first trimester decidua. Decidual NK cells treated with 1,25(OH)(2)D(3) or precursor 25-hydroxyvitamin D(3) (25OHD(3)) for 28 h showed decreased synthesis of cytokines, such as granulocyte-macrophage colony stimulating factor 2 (CSF2), tumor necrosis factor, and interleukin 6, but increased expression of mRNA for the antimicrobial peptide cathelicidin antimicrobial peptide. These data indicate that human decidual cells are able to synthesize active 1,25(OH)(2)D(3), particularly in early gestation, and this may act in an autocrine/paracrine fashion to regulate both acquired and innate immune responses at the fetal-maternal interface.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed2006

Effector activity of decidual CD8+ T lymphocytes in early human pregnancy.

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7-14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR(32)] of 32.7 +/- 5.8) and against K562 target cells (mean SCR(32) of 20.3 +/- 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8(+) T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.

CD8-Positive T-Lymphocytes↗

PubMed2006

Expression of angiogenic growth factors by uterine natural killer cells during early pregnancy.

Remodeling of uterine spiral arteries is critical for the continuation of a successful pregnancy. Uterine natural killer (uNK) cells are the predominant leukocyte population in the early pregnant decidua, and a role for these cells in spiral artery remodeling in pregnancy has been suggested. Angiogenic growth factors were measured in isolated uNK and total (unseparated) decidual cells (8-10 or 12-14 weeks gestation, n=5 each gestational age) after culture for 48 h. Angiopoietin (Ang)1, placental growth factor, transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF)-C were measured by enzyme-linked immunosorbent assay. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. Levels of Ang2, ICAM-1, and KGF, secreted by the total decidual fraction, decreased with increasing gestational age. uNK levels of Ang2 and VEGF-C also decreased with increasing gestational age. At 8-10 weeks gestation, there was no difference in the level of Ang1, Ang2, TGF-beta1, and VEGF-C secreted by uNK cells and the total decidual fraction. At 12-14 weeks, uNK cells secreted significantly lower levels of VEGF-C than the total decidual fraction. Early pregnancy decidua is a major source of angiogenic growth factors whose levels decrease with increasing gestational age, suggesting that they may play a role in spiral artery remodeling. uNK cells appear to be a prominent source of Ang1, Ang2, TGF-beta1, and VEGF-C within the placental bed.

Angiogenic Proteins↗

PubMed2006

Comparison of three multiplex cytokine analysis systems: Luminex, SearchLight and FAST Quant.

Multiplex cytokine analysis technologies have become readily available in the last five years. Two main formats exist: multiplex sandwich ELISA and bead based assays. While these have each been compared to individual ELISAs, there has been no direct comparison between the two formats. We report here the comparison of two multiplex sandwich ELISA procedures (FAST Quant and SearchLight) and a bead based assay (UpState Luminex). All three kits differed from each other for different analytes and there was no clear pattern of one system giving systematically different results than another for any analyte studied. We suggest that each system has merits and several factors including range of analytes available, prospect of development of new analytes, dynamic range of the assay, sensitivity of the assay, cost of equipment, cost of consumables, ease of use and ease of data analysis need to be considered when choosing a system for use. We also suggest that results obtained from different systems cannot be combined.

CD8-Positive T-Lymphocytes↗

PubMed2006

Interferon-gamma inhibits extravillous trophoblast cell invasion by a mechanism that involves both changes in apoptosis and protease levels.

BACKGROUND: Extravillous trophoblast cell (EVT) invasion of decidua and inner third of the myometrium is critical for a successful pregnancy. Many decidual factors are likely to play a role in regulating this process, including uterine natural killer (uNK) cell-derived cytokines. HYPOTHESES: 1) uNK cells are a major source of IFN gamma (IFN-gamma) and 2) IFN-gamma inhibits EVT invasion via an increase in EVT apoptosis and/or a decrease in active protease levels. METHODS: Total decidual and uNK cells from 8-10 wk and 12-14 wk gestational age were cultured. IFN-gamma mRNA (real-time RT-polymerase chain reaction) and protein levels (FastQuant multicytokine analysis) were determined. EVT invasion in the presence of IFN-gamma or anti-IFN-gamma-neutralizing antibodies was assessed. Trophoblast apoptosis and proliferation was assessed in explants by immunohistochemistry for M30 and Ki67. Substrate zymography was performed to determine levels of secreted MMP2, MMP9, and uPA. RESULTS: mRNA and protein for IFN-gamma was detected in both total decidual and uNK cell fractions. Trophoblast invasion was inhibited by IFN-gamma. The level of M30-positive EVT was increased in the presence of IFN-gamma whereas levels of secreted MMP2 were decreased. CONCLUSIONS: uNK cells are a source of IFN-gamma within early human pregnancy decidua. Mechanisms of IFN-gamma inhibition of EVT invasion include both increased EVT apoptosis and reduced levels of active proteases.

Apoptosis↗

PubMed2006

In vivo analysis of trophoblast cell invasion in the human.

In vivo analysis of trophoblast cell invasion is highly dependent on histological techniques, which are amply described in standard textbooks. The emphasis of this chapter therefore lies on material collection and interpretation of tissue sections, rather than on histological techniques per se. Proper identification of vascular structures on placental bed histological sections is important, the more because invading trophoblastic cells induce significant structural changes in uterine blood vessels, which may be disturbed in complicated pregnancies. Guidelines for distinguishing several vascular structures are provided, and different approaches for qualitative and quantitative assessment of spiral artery changes are discussed. The purpose of such studies is not only to obtain a better insight into mechanisms of trophoblast invasion and associated maternal tissue changes, but also to understand placental bed defects in various pregnancy complications.

Arteries↗

PubMed2005

Low oxygen concentrations inhibit trophoblast cell invasion from early gestation placental explants via alterations in levels of the urokinase plasminogen activator system.

Extravillous trophoblast cell (EVT) invasion in early pregnancy occurs in a relatively low-oxygen environment. The role of oxygen in regulation of EVT invasion remains controversial. We hypothesized that 1) culture in 3% oxygen inhibits EVT invasion compared with culture at 8% or 20% oxygen and 2) inhibition of invasion is due to alterations in levels of components of the urokinase plasminogen activator (PLAU, uPA) system rather than through increased apoptosis and/or decreased proliferation. Placental samples (8-10, 12-14, and 16-20 wk gestation) were obtained from women undergoing elective surgical termination of pregnancy or after cesarean section delivery (term) at the Royal Victoria Infirmary, Newcastle upon Tyne, U.K. EVT invasion from placental explants cultured at 3%, 8%, or 20% oxygen was assessed using Matrigel invasion assays. Invasion was assessed on Day 6, explants were harvested for analysis of apoptosis and proliferation, and medium was stored for analysis of PLAU system components by ELISA and casein zymography. Culture at 3% oxygen inhibited EVT invasion. PLAU receptor and plasminogen activator inhibitor-2 protein levels were increased and PLAU activity decreased in these cultures. There was no difference in the proliferation in explants cultured at the three different oxygen concentrations. Apoptosis, assessed by M30 immunostaining, was increased in EVT at both 3% and 8% oxygen. The reduction in the invasive capacity of EVT cultured at 3% oxygen appears to be mediated both by a general inhibition of the PLAU system and a decrease in the number of cells available to invade.

Amyloid beta-Protein Precursor↗

PubMed2005

Inhibition of trophoblast cell invasion by TGFB1, 2, and 3 is associated with a decrease in active proteases.

Invasion of extravillous trophoblast cells into the uterus in human pregnancy is tightly regulated. The transforming growth factor-beta (TGFB) family has been suggested to play a role in controlling this process. We hypothesized that TGFB1, 2, and 3 would inhibit the invasive capacity of extravillous trophoblast cells. We also studied trophoblast apoptosis and proliferation and secreted protease levels as potential mechanisms by which these cytokines may act. Inhibition of endogenous TGFB1, 2, and 3 with neutralizing antibodies increased the invasive capacity of extravillous trophoblast cells derived from placental explants. Similarly, addition of exogenous TGFB1, 2, and 3 inhibited the invasive capacity of these cells in a dose-dependent manner. Proliferation of trophoblast in the placental explants did not alter in response to any of the cytokines tested. Apoptosis of villous and extravillous trophoblast did not alter in response to TGFB1, 2, and 3. There was a reduction in secreted levels of matrix metalloproteinase (MMP) 9 and urokinase plasminogen activator in response to all three cytokines. MMP2 and tissue inhibitor of metalloproteinase 1 and 3 levels were not altered. These results suggest that TGFB1, 2, and 3 inhibit trophoblast invasion by a mechanism dependent on reduced protease activity.

Apoptosis↗

PubMed2005

Neonatal measles immunity in rural Kenya: the influence of HIV and placental malaria infections on placental transfer of antibodies and levels of antibody in maternal and cord serum samples.

INTRODUCTION: Young infants are protected from measles infection by maternal measles antibodies. The level of these antibodies at birth depends on the level of antibodies in the mother and the extent of placental transfer. We investigated predictors of levels of measles antibodies in newborns in rural Kenya. METHODS: A total of 747 paired maternal-cord serum samples (91 from human immunodeficiency virus [HIV]-infected and 656 from HIV-uninfected mothers) were tested for measles immunoglobulin G antibodies. Placental malaria infection was determined by biopsy. Data on pregnancy history, gestational age, and anthropometric and socioeconomic status were collected. RESULTS: Infants born to HIV-infected mothers were more likely (odds ratio, 4.6 [95% confidence interval {CI}, 2.2-9.7]) to be seronegative and had 35.1% (95% CI, 9.8%-53.2%) lower levels of measles antibodies than did those born to HIV-uninfected mothers. Preterm delivery, early maternal age, and ethnic group were also associated with reduced levels of measles antibodies. There was little evidence that placental malaria infection was associated with levels of measles antibodies in newborns. CONCLUSION: Our results suggest that maternal HIV infection may reduce levels of measles antibodies in newborns. Low levels of measles antibodies at birth render children susceptible to measles infection at an early age. This is of concern in sub-Saharan African countries, where not only is the prevalence of HIV high, but measles is the cause of much morbidity and mortality.

Adolescent↗

PubMed2005

Macrophages and not granulocytes are involved in cervical ripening.

To clarify the role of leucocytes in human cervical ripening and dilatation, cervical biopsies were obtained from six non-pregnant women, eight women undergoing early termination of pregnancy, 18 pregnant women undergoing elective Caesarean section at term (both with and without a ripe cervix as determined by Bishop score) and 11 women after term vaginal delivery. Leucocytes were localised by immunohistochemistry labelling and quantified in subepithelial and deep stromal areas. CD45+ leucocytes were more numerous in the subepithelial area of the cervix than in the deep stroma in all groups (P<0.01). CD14+ macrophages and CD15+ granulocytes were increased in both the subepithelial and deep stromal areas only in the vaginal delivery group (P<0.01). The number of macrophages in the ripening cervix (Bishop score above 4) was higher than in the unripe cervix (Bishop score 4 or less; P<0.05) with no differences in other leucocyte populations. CD3+ CD8+ T cells in the subepithelial area were reduced in late pregnancy and after vaginal delivery (P<0.01), but showed no relationship to Bishop score. Macrophages and granulocytes may be involved in the process of cervical dilatation, but macrophage infiltration into the ripening cervix before labour suggests their role in the ripening process. Reduced numbers of CD3+ CD8+ T-lymphocytes in late pregnancy and after vaginal delivery suggests that local immunity is down-regulated in the late pregnancy period. Regional differences in leucocyte subpopulations in the cervix indicate that leucocyte infiltration is likely to be regulated by local factors.

Antigens, CD↗

PubMed2005

Human uterine natural killer cells: a reappraisal.

The presence of granulated cells within the uterus of many species has been recognised for many years but only recently have these been recognised to be a type of NK cell. Various terms have been applied to the cells, including endometrial granulocyte, K cell and, in mouse and rat, granulated metrial gland cell. Although early studies are often based on histology and electron microscopy, these often include important information for current studies. In vitro studies of purified cells have focused particularly on cytotoxicity and cytokine production and roles in the control of trophoblast invasion and spiral artery remodelling in human pregnancy have been proposed. Evidence in mouse has implicated uNK cell production of IFN-gamma in vascular remodelling but evidence for such a role for human uNK cells remains to be established. Investigation of uNK cells in human pregnancy is hampered by the lack of availability of tissues from the first half of the second trimester of pregnancy when vascular remodelling occurs and also by possible differences between cells from different regions of decidua. The presence of similar cells in species with no trophoblast invasion into the uterus and epitheliochorial placentation raises the question of whether control of trophoblast invasion by human uNK cells is important in vivo and raises the possibility of another function which is conserved between species.

Animals↗

PubMed2005

Maternal vitamin A supplementation and immunity to malaria in pregnancy in Ghanaian primigravids.

BACKGROUND: Vitamin A supplementation is believed to enhance immune responses to infection but few studies have assessed its effects on anti-malarial immunity, especially during pregnancy when women are at increased risk from both vitamin A deficiency and pregnancy-associated malaria. The pathological effects of malaria in pregnancy are believed to be due to the sequestration of parasites in the placenta mediated via binding of variant surface antigens (VSA) expressed on the surface of P. falciparum infected red blood cells to placental chondroitin sulphate A (CSA). METHODS: We conducted a randomized double-blind controlled trial of vitamin A supplementation in 98 primigravid Ghanaian women to investigate the effects of vitamin A supplementation on levels of IgG antibodies binding to VSA of a clinical, P. falciparum placental isolate and to two isolates selected (or not) for adherence to CSA in vitro (anti-VSACSA IgG or anti-VSA IgG). Placental malarial infection was determined by placental blood smear and histology. RESULTS: Vitamin A supplementation was non-significantly associated with a decreased risk of active or chronic-active placental malarial infection compared to past, resolved infection at delivery, as determined by histology (OR=0.42, P=0.13--adjusted for level of education). After adjustment for differences in baseline values, levels of anti-VSACSA IgG to a placental, CSA-adherent isolate (EJ-24) but not to two isolates selected for CSA-adhesion in vitro (FCR3CSA and BusuaCSA), were significantly lower in women receiving vitamin A supplementation than in women receiving placebo (P=0.002). There was no apparent effect of vitamin A supplementation to levels of Ab to non-CSA-adherent parasite isolates. CONCLUSIONS: The data suggest that the reduction in the levels of anti-VSACSA antibodies to the known placental malaria isolate may reflect reduced intensity or duration of placental parasitaemia in women receiving vitamin A supplementation. These observations are of potential public health significance and deserve further investigation.

Adult↗

PubMed2005

Rapid acquisition of isolate-specific antibodies to chondroitin sulfate A-adherent plasmodium falciparum isolates in Ghanaian primigravidae.

Recent evidence suggests that pregnancy-associated malaria (PAM), associated with maternal anemia and low birth weight, results from preferential sequestration of parasitized red blood cells (pRBC) in the placenta via binding of variant surface antigens (VSA) expressed on the surface of pRBC to chondroitin sulfate A (CSA). The VSA mediating CSA binding (VSA(CSA)) and thus sequestration of pRBC in the placenta are antigenically distinct from those that mediate pRBC sequestration elsewhere in the body, and it has been suggested that VSA(CSA) are relatively conserved and may thus constitute an attractive target for vaccination against PAM. Using flow cytometry, levels of antibody to VSA and VSA(CSA) expressed on the surface of red blood cells infected with Plasmodium falciparum isolates were measured during pregnancy and lactation in Ghanaian primigravid women enrolled in a trial of maternal vitamin A supplementation. Antibody responses to VSA(CSA) were detected within the first trimester of pregnancy and increased with increasing duration of pregnancy, and they seemed to be isolate specific, indicating that different CSA-adherent parasite lines express antigenically distinct VSA and thus may not be as antigenically conserved as has been previously suggested. Levels of anti-VSA(CSA) were not significantly associated with placental malarial infection determined by histology, indicating that primary immune responses to VSA(CSA) may not be sufficient to eradicate placental parasitemia in primigravidae.

Animals↗

PubMed2004

Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria.

BACKGROUND: Pregnancy-associated malaria caused by Plasmodium falciparum adherence to chondroitin sulfate A in the placental intervillous space is a major cause of low birthweight and maternal anaemia in areas of endemic P falciparum transmission. Adhesion-blocking antibodies that specifically recognise parasite-encoded variant surface antigens (VSA) are associated with resistance to pregnancy-associated malaria. We looked for a possible relation between VSA-specific antibody concentrations, placental infection, and protection from low birthweight and maternal anaemia. METHODS: We used flow cytometry to measure VSA-specific IgG concentrations in plasma samples taken during child birth from 477 Kenyan women selected from a cohort of 910 women on the basis of HIV-1 status, gravidity, and placental histology. We measured VSA expressed by one placental P falciparum isolate and two isolates selected or not selected for chondroitin sulfate A adhesiveness in-vitro. FINDINGS: Concentrations of plasma IgG specific for VSA, expressed by chondroitin sulfate A-adhering parasites (VSA in pregnancy-associated malaria or vsa-pam), increased with gravidity and were associated with placental histological findings. Women with chronic pregnancy-associated malaria and low or absent VSA-PAM-specific IgG had lower haemoglobin values (reduced by 17 g/L; 95% CI 8.1-25.2) and delivered smaller babies (birthweight reduced by 0.26 kg; 0.10-0.55) than did corresponding women with high VSA-PAM-specific IgG. No such relation was shown for concentrations of IgG with specificity for non-pregnancy-associated malaria VSA. INTERPRETATION: VSA-PAM-specific IgG protects against low birthweight and maternal anaemia. Our data indicate an important mechanism of clinical protection against malaria and raise hope for the clinical effectiveness of a potential VSA-based vaccine against pregnancy-associated malaria.

Animals↗

PubMed2004

Vitamin D and placental-decidual function.

The active form of vitamin D (1,25-dihydroxyvitamin D(3), 1,25[OH](2)D(3)) has well-established effects on bone metabolism and mineral homeostasis. However, recently it has become clear that 1,25(OH)(2)D(3) has potent antiproliferative and immunomodulatory actions that are not immediately linked to its role as a skeletal regulator. Both the nuclear receptor for 1,25(OH)(2)D(3) (vitamin D receptor, VDR) and the vitamin D-activating enzyme 1alpha-hydroxylase are expressed in a wide variety of nonclassic tissues, highlighting the potential for local autocrine-paracrine responses rather than traditional endocrine effects. Prominent among the tissues that express 1alpha-hydroxylase is the placenta-decidua, and this has raised important questions concerning the potential role of locally generated 1,25(OH)(2)D(3) as a modulator of fetal-placental development and function. When bound to the VDR, 1,25(OH)(2)D(3) regulates key target genes associated with implantation, such as HOXA10, whereas the potent immunosuppressive effects of 1,25(OH)(2)D(3) suggest a role in implantation tolerance. These observations are further supported by data from our group showing increased expression of 1alpha-hydroxylase and VDR in first-trimester trophoblast and decidua from human pregnancies. Studies by other groups have reported abnormal expression of 1alpha-hydroxylase in preeclamptic pregnancies, revealing a potential role for 1,25(OH)(2)D(3) as a regulator of placentation. The effect of vitamin D on reproduction has been further endorsed by murine gene knockout models for 1alpha-hydroxylase and VDR, both of which are infertile. These observations and others are discussed in this article in which we postulate an active role for 1,25(OH)(2)D(3) in placenta-decidua. In particular, we describe how induction of the vitamin D-activating enzyme 1alpha-hydroxylase in early gestation might provide a mechanism by which environmental or dietary vitamin D can influence fetal-placental development.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed2004

Increased expression of 25-hydroxyvitamin D-1alpha-hydroxylase in dysgerminomas: a novel form of humoral hypercalcemia of malignancy.

Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic disorder usually associated with increased synthesis of parathyroid hormone-related peptide (PTHrP). Unlike non-cancer forms of hypercalcemia, HHM does not routinely involve increased circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Dysgerminomas are a notable exception to this rule, previous reports having described hypercalcemia with elevated serum 1,25(OH)2D3. To investigate the etiology of this form of HHM we have characterized expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)2D3, 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase), in a collection of 12 dysgerminomas. RT-PCR analyses indicated that mRNA for 1alpha-hydroxylase was increased 222-fold in dysgerminomas compared to non-tumor ovarian tissue. Parallel enzyme assays in tissue homogenates showed that dysgerminomas produced fivefold higher levels of 1,25(OH)2D3 compared to normal ovarian tissue. Immunolocalization studies indicated that 1alpha-hydroxylase was expressed by both tumor cells and by macrophages within the inflammatory cell infiltrate associated with dysgerminomas. The immunological nature of the increased 1,25(OH)2D3 production observed in dysgerminomas was further emphasized by correlation between expression of 1alpha-hydroxylase and the endotoxin recognition factors CD14 and toll-like receptor 4 (TLR4). These data suggest that inflammatory mechanisms associated with dysgerminomas are the underlying cause of the increased expression and activity of 1alpha-hydroxylase associated with these tumors. We further postulate that this autocrine/paracrine action of 1alpha-hydroxylase may lead to increased circulating levels of 1,25(OH)2D3 and a form of HHM which is distinct from that seen with PTHrP-secreting tumors.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed2003

HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR.

OBJECTIVE: To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin. METHODS: Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR. RESULTS: All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers. CONCLUSIONS: These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.

Adult↗

PubMed2002

The ontogeny of 25-hydroxyvitamin D(3) 1alpha-hydroxylase expression in human placenta and decidua.

In addition to its classical calciotropic effects, the active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a potent anti-proliferative/immunomodulatory secosteroid. The enzyme that catalyzes the synthesis of 1,25(OH)(2)D(3), 1alpha-hydroxylase (1alpha-OHase), is expressed in many human tissues, highlighting its possible role as an autocrine/paracrine activator of vitamin D. Immunohistochemical and RNA analyses were used to characterize the ontogeny of 1alpha-OHase expression in human placenta and decidua. Protein for 1alpha-OHase was detectable in trophoblast and decidua; the latter being stronger in decidualized stromal cells than macrophages, with no staining of lymphocytes. Quantitative reverse transcriptase-polymerase chain reaction was used to assess changes in mRNA expression for 1alpha-OHase at different gestations: first (mean, 9.1 +/- 1.5 weeks); second (mean, 14 +/- 1.8 weeks), and third trimester (mean, 39.3 +/- 2.5 weeks). 1alpha-OHase expression in decidua was approximately 1000-fold higher in first (95% confidence limits, 611 to 1376) and second (95% confidence limits, 633 to 1623) trimester biopsies when compared with the third trimester (95% confidence limits, 0.36 to 2.81) (both P < 0.001). In placenta, 1alpha-OHase expression was 80-fold higher in the first (range, 42 to 137) and second (range, 30 to 199) trimester when compared with third trimester biopsies (0.6 to 1.6) (both P < 0.001). Similar results were obtained by semiquantitative IHC. Parallel analysis of the receptor for 1,25(OH)(2)D(3) (vitamin D receptor) indicated that, as with 1alpha-OHase, highest levels of expression occurred in first trimester decidua. However, changes in vitamin D receptor mRNA expression across gestation were less pronounced than 1alpha-OHase. These spatiotemporal data emphasize the potential importance of 1alpha-OHase during early fetoplacental life and, in particular, suggest an autocrine/paracrine immunomodulatory function for the enzyme.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗