Polycyclic aromatic hydrocarbons in placenta, maternal blood, umbilical cord blood and milk of Indian women

Human placenta, umbilical cord blood, maternal blood and breast milk samples from mothers were analysed for the presence of selected polycyclic aromatic hydrocarbons (PAHs). Benzo(a) pyrene (B(a)P), dibenzo(a,c)anthracene (DBA) and chrysene (Chy) were detected in all the four types of sample. Levels of dibenzo(a,c)anthracene were higher in the above samples compared with the other two PAHs. Umbilical cord blood and breast milk samples showed relatively high concentrations of all the three PAHs and thus demonstrated that the developing foetus/new born were exposed to these carcinogenic environmental contaminants. The possible implications of PAHs in relation to human health are discussed.     

Vasodilator actions of urocortin and related peptides in the human perfused placenta in vitro

Urocortin, is a recently isolated peptide belonging to the CRH family that binds with high affinity to the CRH2 receptor. Like CRH, urocortin causes hypotension in the rat, but its vasoactive actions have not yet been studied in the human. We have compared the vasoactive properties of urocortin, CRH, and urotensin-1 in the human fetal placental vasculature in vitro. Single placental lobules were bilaterally perfused (maternal and fetal sides, 5 mL/min each; 95% O2-5% CO2; 37 C), and changes in fetal arterial perfusion pressure were recorded. Submaximal vasoconstriction was induced by PGF2alpha (4+/-0.7 micromol/L), which increased perfusion pressure from 19.6+/-1.4 to 100.7+/-3.1 mm Hg (n=38; P < 0.001). Subsequent fetal arterial infusion of urocortin (0.001-1 nmol/L) caused concentration-dependent vasodilatation. Urocortin was equipotent with urotensin-1 and 25 times more potent than CRH in causing vasodilatation. Nevertheless, the maximum vasodilator responses to each of the peptides were similar (P > 0.05). The CRH receptor antagonist, alpha-helical CRH-(9-41) (0.2 nmol/L) significantly attenuated the vasodilatation produced by urocortin, urotensin-1, and CRH (P < 0.05). These results indicate a possible physiological role for urocortin in the modulation of human fetal placental vascular tone by activation of CRH2-like receptors.     

Nitric oxide regulation of corticotropin-releasing hormone release from the human perfused placenta in vitro

We have investigated the regulatory role of nitric oxide (NO) in corticotropin-releasing hormone (CRH) release from the human perfused placental lobule in vitro. The effects of the NO donor sodium nitroprusside, the NO synthase inhibitor N omega-nitro-L-arginine, and the NO substrate L-arginine on human (h) placental CRH secretion have been studied. Single lobules of term placentae were bilaterally perfused with Krebs solution (5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3). Fetal and maternal perfusates were collected at 4 C every 30 min for 3 h. CRH immunoreactivity (CRH-IR) in perfusates was measured by RIA using the 41-residue synthetic CRH as standard, 125I-labeled Tyr-hCRH as tracer, and a rabbit anti-CRH antibody Y2BO. The sensitivity of the assay was 0.13 pmol/L. Under basal conditions, human perfused placentae in vitro continuously secreted CRH-IR, which diluted in parallel to a synthetic hCRH-(1-41) standard curve. Size-exclusion chromatography of placental perfusates using a Sephadex G-50 column indicated that placental CRH-IR predominately coeluted with hCRH-(1-41) standard. Basal maternal perfusate CRH-IR levels (27 +/- 4 pmol/L) released from perfused placental lobules were nearly 10-fold greater than fetal perfusate CRH-IR levels (3.4 +/- 0.7 pmol/L; P < 0.05). Infusion of sodium nitroprusside (30-100 mumol/L) into the maternal and fetal placental circulations inhibited CRH-IR release into maternal perfusate in a concentration-dependent manner, but did not inhibit CRH-IR release into the fetal perfusate. N omega-nitro-L-arginine (100 mumol/L) increased placental CRH-IR secretion into fetal perfusate, and this effect was reversed by the infusion of L-arginine (100 mumol/L), which also reduced release below basal levels. In contrast, maternal perfusate CRH-IR levels were not affected by N omega-nitro-L-arginine or L-arginine. These results indicate that the human perfused placenta in vitro releases a substance of similar mol wt and hCRH-IR. Moreover, modulators of the NO signaling pathway differentially affect placental secretion of CRH-IR into the maternal and fetal perfusates. These data are consistent with the involvement of NO in the regulation of placental CRH release during pregnancy.     

Effect of certain drugs in perfused human placenta XII: autacoid antagonism by phenothiazines

The effects of chlorpromazine, prochlorperazine, and trifluoperazine on the pressor actions of serotonin, angiotensin, and bradykinin in the perfused vessels of full-term human placentas were investigated. A significant inhibition of the effect of serotonin was observed with trifluoperazine and chlorpromazine but not with prochlorperazine. The inhibition is attributed to the ability of phenothiazines to cause adrenergic blockade. Because both bradykinin and angiotensin could not be consistently antagonized, it is concluded that they must act primarily via musculotropic mechanisms and only secondarily by stimulation of adrenergic receptors.     

Factors influencing pulmonary surfactant protein A levels in cord blood, maternal blood and amniotic fluid

The sequence encoding the truncated core protein (amino acids 1-98) of hepatitis C virus (HCc) was expressed in E. coli for production of HCc(1-98), or fused with the truncated core antigen (HBcAg) and segments from the preS1 and preS2 regions from hepatitis B virus (HBV) for production of HBcPreS1PreS2HCc(1-98). The HCc(1-98) and HBcPreS1PreS2HCc(1-98) proteins reacted with sera from HCV-infected individuals by immunoblot analyses, while the latter protein also exhibited HBV core antigenicity. They induced antibodies against HBcAg and/or HCV core protein in rabbits and in mice. Moreover, HBcPreS1PreS2HCc(1-98) is more immunogenic than HCc(1-98) in terms of anti-HCc induction. An ELISA that employed recombinant HCV core antigens of either HCc(1-98) or HBcPreS1PreS2HCc(1-98) to detect anti-HCc and/or anti-HBc antibodies was developed. Evaluation of serum samples with different status of HBV and HCV infections suggested that HCc(1-98) might be suitable for the determination of antibodies against HCV core protein, while HBcPreS1PreS2HCc(1-98) might be of value to detect HCV and/or HBV infection in donated blood in HBV low-prevalence countries.     

Immunohistochemical localization of carboxypeptidases E and D in the human placenta and umbilical cord

Carboxypeptidase E (CPE) is highly concentrated in neuroendocrine tissues and is the only carboxypeptidase detected in mature secretory vesicles. Carboxypeptidase D (CPD), a carboxypeptidase with CPE-like activity, is widely distributed in tissues and is present in the trans-Golgi network. Previous work had shown that both CPE and CPD are expressed in the human placenta and that CPD is expressed at much higher levels than CPE. The present work provides evidence for the co-localization of CPE and CPD to basal plate extravillous trophoblasts and maternal uteroplacental vascular endothelial cells, chorionic villous endothelial cells, amnionic epithelial cells, and umbilical venous and arterial smooth muscle cells. Whereas the intensity of CPD immunostaining is similar in the placenta and umbilical cord, CPE staining in the placenta is much weaker than in the umbilical cord, suggesting that CPD plays a more important role in the processing of placental peptides. Immunoelectron microscopy of umbilical venous smooth muscle cells shows subcellular localization of both enzymes to the rough endoplasmic reticulum. In addition, CPE is present just subjacent to the cell membrane. The difference in cellular and subcellular localization between the two enzymes indicates that they perform distinct functions in the processing of placental peptides and proteins.     

Lack of effect of cocaine on lysine and alanine uptake in human placental villi or transfer in perfused human placenta

The effect of cocaine on lysine and alanine uptake in human placental villi and transfer across the dually perfused placenta was studied. Uptake (in terms of the intracellular to extracellular distribution ratio) of alanine and lysine was 2.81 +/- 0.30 (n = 5) and 1.45 +/- 0.24 (n = 5) respectively and was unaffected by cocaine (50-500 ng mL(-1) in the incubation medium. In the dually perfused placenta, the clearance index (ratio of amino acid to antipyrine clearance) was 0.35 +/- 0.03 and 0.30 +/- 0.05 and the transfer index (ratio of amino acid to L-glucose clearance) was 2.20 +/- 0.07 and 1.89 +/- 0.29 for lysine and alanine respectively. Cocaine at concentrations of 100 ng mL(-1) or 250 ng mL(-1) had no effect on the clearance of either amino acid. The results of this study indicate that concentrations of cocaine likely to be encountered in vivo do not affect uptake of lysine or alanine by placental villi or transfer across the perfused placental lobule, in contrast with the report that cocaine reduces uptake of alanine by placental vesicles. Experimental models must be critically evaluated before accepting the results as pertinent to a clinical situation.     

Identification of a unique form of p53 in human cord blood associated with the development of maternal autoantibodies

PROBLEM: The possible link between p53-reactive antibodies in multiparous women and exposure to a unique p53 protein during pregnancy was examined. METHOD OF STUDY: p53-reactive antibodies were evaluated in sera from nulligravid and multiparous women and patients with ovarian cancer by Western immunoblot. Furthermore, the presence of p53 protein was assayed in cord blood by enzyme-linked immunosorbent assay. Cord blood-derived p53 was compared structurally by protein fingerprinting and functionally by gel mobility shift assay to other isolates of p53. RESULTS: Antibodies reactive with wild-type p53 were observed in 92% of multiparous women and 42% were reactive with one tumor-derived p53 protein. p53 protein was detected in 27 of 154 samples of cord blood. Structural analysis indicated that the fetal p53 resembled the UL-1 p53. Functionally, the fetal and UL-1 protein failed to bind DNA. CONCLUSIONS: Fetal p53 protein seems to be distinct from wild-type p53, characterized by enhanced stability, structural differences and inability to bind DNA, analogous to alternatively spliced variants. Exposure to fetal p53 protein may form the basis for immunologic protection against cancer induced by multiparity.     

A highly sensitive polymerase chain reaction method reveals the ubiquitous presence of maternal cells in human umbilical cord blood

Umbilical cord blood is considered an alternate source of hematopoietic stem cells in bone marrow transplantation. However, its use might be hampered by contamination of neonatal blood with maternal cells, which could contribute unacceptably to graft-vs.-host disease (GVHD) after transplant. In a previous study (Socié et al., Blood 83:340, 1994), we used polymerase chain reaction (PCR) amplification of minisatellite sequences (sensitivity 1-0.1%) to address the question of this contamination. In a single case among 47 analyzed, we were able to detect a maternal-specific allele in the cord blood sample. We have now studied the same cord samples using a highly sensitive, allele-specific PCR amplification method. A maternal allele could be discriminated from neonate alleles in 10 cases and maternal cells were detected in all 10 cord blood samples. These cells amounted to 10(-4) to 10(-5) of cord blood nucleated cells. In three cases, cord blood separated cell subpopulations could be analyzed and were found to contain maternal cells at about the same level. The presence of maternal cells at such a low level in cord blood samples probably would have no effect on GVHD in a clinical setting of transplantation but raises interesting questions in terms of materno-fetal immune tolerance and transmission of viruses (in particular human immunodeficiency virus) from infected mother to child.     

Simultaneous determination of spironolactone and its metabolites in human plasma

This study describes a specific, precise, sensitive and accurate method for determination of unchanged spironolactone and its major active metabolites in human plasma. After one-step liquid-liquid extraction, analysis of the parent drug and its metabolites was performed in one chromatographic run, using a high performance liquid chromatography (HPLC) method with a programmed switchover of the UV wavelength. Spironolactone and 7 alpha-thiomethyl-spironolactone were detected at 245 nm, while canrenone and internal standard were detected at 280 nm. The column used was an S5 ODS2 (500 mm x 4.6 mm i.d.). The mobile phase was a mixture of acetonitrile-aqueous orthophosphoric acid (pH 3.4). Chromatographic separations were performed at 5 degrees C. The standard curves were linear over the range 10-400 ng ml-1 for spironolactone and 10-600 ng ml-1 for 7 alpha-thiomethyl-spironolactone and canrenone. The precision and accuracy of the method were confirmed by relative standard deviations below 10% for different concentrations, except for the concentration equal to the quantitation limit, where these parameters ranged from 12-15%. The recovery was above 80% for all investigated compounds and for the internal standard. The assay proved to be suitable for pharmacokinetic studies of spironolactone.     

Autacoid interactions in the regulation of blood flow in the human placenta

1. Interactions between autacoids may play important roles in the regulation of blood flow in the foetal placenta. In order to investigate this aspect of placental haemodynamics, human normal-term placentae were perfused in vitro and the responses of the foetal vessels to various combinations of vasoactive agents were determined. 2. Vasoconstriction responses to 5-hydroxytryptamine (5-HT) were potentiated in the presence of endothelin-1 (ET-1), the thromboxane A2-mimetic U46619 and a nitric oxide synthase inhibitor, N-nitro-L-arginine (NOLA), but not in the presence of angiotensin II. 3. N-Nitro-L-arginine caused vasoconstriction of the perfused placenta and indomethacin attenuated this effect and blocked the potentiation of the 5-HT response by NOLA. 4. Indomethacin did not affect ET-1-induced pressure increases and infusion of U46619 had no effect on release of ET-like immunoreactivity into the foetal placental circulation. 5. The present study provides evidence of interactions between several autacoids in human perfused placentae in vitro. These interactions may play important roles in foetal placental haemodynamics in normal or pathological situations.     

Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood

Umbilical cord blood (UCB) and mobilized peripheral blood (MPB) provide an alternate source to bone marrow for transplantation. Expansion in vitro of stem/progenitor cell populations from these sources may provide adult-sized grafts otherwise not attainable because of the limited cell numbers available in the case of UCB or because of numerous rounds of apheresis required for sufficient MPB cells. We asked whether continuous perfusion culture could be employed in ex vivo expansion to produce clinically relevant numbers of stem/progenitor cells from these sources. To evaluate MPB, 1-10 million leukocytes, from patients who had received either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF), were inoculated into bioreactors, with or without irradiated, allogeneic stroma. The growth factor combination in the perfusion medium consisted of interleukin-3 (IL-3), stem cell factor (SCF), GM-CSF and erythropoietin (Epo). Under the best conditions tested, total cell numbers, granulocyte-macrophage colony-forming units (CFU-GM), and long-term culture-initiating cell (LTC-IC) populations were expanded by about 50-, 80-, and 20-fold, respectively, over 14 days. At low cell inocula (1 million), the presence of stroma enhanced the expansion of total cells and CFU-GM but not of LTC-IC. When SCF was not included in the medium, both total cells and CFU-GM expanded to a much lesser extent, but again the expansion of LTC-IC was not affected. At the higher cell inoculum (10 million), expansions of total cells and CFU-GM were equivalent with or without stroma. To evaluate UCB, cells were placed into bioreactors with or without irradiated, allogeneic stroma, and the bioreactors were perfused with medium containing the four standard growth factors. After 6-14 days, in several independent experiments, 20-24 million cells were harvested from bioreactors perfused with SCF-containing medium, irrespective of the presence or absence of preformed stroma. Similarly, in reactors perfused with SCF-containing medium (with or without stroma), an average 40- to 60-fold expansion of CFU-GM was obtained, yielding an average of 1.5-1.8 x 10(5) CFU-GM per reactor. Harvested cells were thus up to 40-fold enriched in CFU-GM in comparison to the inoculum. In the absence of SCF, cell expansions averaged 1.5- to 2-fold, and CFU-GM were expanded only 10- to 14-fold by day 14. As before, the presence of preformed stroma did not affect either cell or CFU-GM yields, provided the cell inoculum was at least 4.5 million cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies

1. It has been suggested that a deficiency of nitric oxide (NO) may explain many of the pathophysiological features of pre-eclampsia (PE) and intra-uterine (foetal) growth retardation (IUGR). To elucidate further the role of NO in the pathophysiology of pregnancy we have determined the relative amount and activity of NO synthase (NOS) in first trimester and normal-term placental tissues, as well as in the placenta and umbilical cord in pregnancies complicated by PE and IUGR, using NG-nitro-L-[2,3,4,5(-3)H]-arginine ([3H]-L-NOARG) binding, quantitative in vitro autoradiography, [3H]-arginine to [3H]-citrulline conversion and Western blotting. 2. Specific, high affinity (KD = 38 nM) [3H]-L-NOARG binding was demonstrated in the villous trophoblast of normal-term placentae. Binding was calcium-independent, stereoselective and exhibited a rank order of inhibition by NOS inhibitors and substrate (L-NOARG > or = L-NMMA > or = 7-NI > L-NAME > L-Arg > or = L-NIO > ADMA). 3. [3H]-L-NOARG binding density and NOS activity were both significantly greater in placental tissues from first trimester and PE or IUGR complicated pregnancies compared to normal-term placentae. 4. Western blotting, using an endothelial NOS peptide antiserum, demonstrated a approximately 140 KDa protein band in placental extracts and indicated that the amount of immunoreactive material was significantly greater in first trimester compared to normal-term placentae. 5. Specific [3H]-L-NOARG binding was also localized to the endothelial lining of umbilical arteries and veins, binding density being greater in the artery than the vein. [3H]-L-NOARG binding to the umbilical artery endothelium was significantly lower in PE and IUGR complicated pregnancies compared to normal-term controls. 6. The role of trophoblast-derived NO in human placental pathophysiology remains to be established, but differences in the amount of placental [3H]-L-NOARG binding, NOS activity and immunoreactive material indicate that expression of NOS in the villous trophoblast falls during pregnancy. Conversely, the apparent reduction in NOS in the umbilical artery endothelium in PE and IUGR complicated pregnancies may be indicative of endothelial dysfunction.     

The association between maternal cocaine use and placenta previa

We compared various diagnostic tests for their abilities to detect Mycobacterium ulcerans infection in specimens from patients with clinically active disease. Specimens from 10 patients from the area of Zangnanado (Department of Zou, Benin) with advanced, ulcerated active M. ulcerans infections were studied by direct smear, histopathology, culture, PCR, and oligonucleotide-specific capture plate hybridization (OSCPH). A total of 27 specimens, including 12 swabs of exudate collected before debridement and 15 fragments of tissue obtained during debridement, were submitted to bacteriologic and histopathologic analysis. The histopathologic evaluation of tissues from all six patients so tested revealed changes typical of those caused by M. ulcerans infection. Five specimens were contaminated, and M. ulcerans was cultivated on L?wenstein-Jensen medium from 12 of the remaining 22 (54.5%) specimens. Detection of mycobacteria was performed by PCR, and M. ulcerans was detected by OSCPH with a new probe (5'-CACGGGATTCATGTCCTGT-3') reacting with M. ulcerans and Mycobacterium marinum. In 10 of 22 (45.5%) specimens, M. ulcerans was identified by PCR-OSCPH. There was no statistically significant difference between the detection of M. ulcerans by culture and by PCR-OSCPH (P > 0.05). This is the first demonstration of an amplification system (PCR-OSCPH) with a sensitivity similar to that of culture for the direct and rapid recognition of M. ulcerans in clinical specimens. This system is capable of identifying M. ulcerans, even in paucibacillary lesions. Our findings suggest that PCR-OSCPH should be used in the quest for the elusive environmental reservoir(s) of M. ulcerans.     

Transport of aspirin and its metabolites through human erythrocyte membrane

A 72-year-old man developed a very progressive neuropsychologic deficit 6 years ago, beginning with isolated visual topographic memory disturbances and visuo-constructive apraxia without additional manifestations of dementia. The syndrome worsened thereafter with the emergence of visual agnosia, simultagnosia, psychic paralysis of gaze, auditivo-verbal agnosia, and recently an amnestic syndrome with confabulation and confusion (at the end of 1989). CT scans, which remained unchanged over the years, showed mild, focal atrophic changes revealed by dilatation of the right occipital horn. His angiograms were normal. Two SPECT studies (with HMPAO measurements), performed 6 years from the onset, detected marked hypoperfusion in both parieto-occipital regions, mainly on the right side. Progressive focal degenerative disease without dementia is a relatively new syndrome, especially in cases with progressive aphasia. As noted in our patient, progressive disturbances initially localized in the right parieto-occipital region followed by posterior bilobar degeneration (pronounced on the right side) without dementia until late in the course may represent another exceptionally reported characteristic of this new syndrome. It is suggested that this variant of the Mesulam syndrome is more likely explained by progressive atrophy of the Alzheimer type.     

Blastogenic response of activated human umbilical cord blood T-lymphocytes

Donor T-lymphocytes are thought to play a crucial role in both acute and chronic graft-versus-host disease (GvHD), pathological conditions that frequently complicate allogeneic bone marrow transplantation. These diseases are described as occurring with a lower incidence and lesser severity when human umbilical cord blood (HUCB) cells, which have recently emerged as a potential source of hematopoietic progenitors, are used for transplantation. This condition is probably related to the immaturity of neonatal HUCB T cells. Lymphocyte blastogenic response to phytohemagglutinin (PHA), evaluated by means of flow cytometry, is a useful tool for testing the functional ability of T-cells to display an immune response against allo-antigens, reproducing in vitro the in vivo mechanism of activation. This study was designed to verify whether an impairment in HUCB T-cell ability to undergo an in vitro blastogenic response to mitogens could explain their reduced in vivo ability to induce GvHD.     

Partitioning of dioxins, dibenzofurans, and coplanar PCBS in blood, milk, adipose tissue, placenta and cord blood from five American women

Partitioning of dioxins, dibenzofurans and the dioxin-like coplanar PCBs was determined by congener-specific high resolution gc-ms analysis of compounds in 6 tissue samples each from 5 women. Samples were whole blood obtained prior to delivery; maternal adipose tissue, cord blood and placenta obtained during cesarean section delivery; and whole blood and milk taken at the time of first obstetrical follow-up examination, one to two months following delivery. All women lived in upstate New York. Specimens were collected in late 1995 and early 1996. Mean measured levels of total PCDDs, PCDFs and coplanar PCBs were 352 pg/g for adipose tissue, 526 pg/g for predelivery blood, 182 pg/g for placenta, 165 pg/g for cord blood, 352 pg/g for postpartum blood and 220 pg/g for milk. Mean total TEQ levels were 11.6 pg/g TEQ for adipose tissue, 12.1 pg/g TEQ for predelivery blood, 10.5 pg/g TEQ for placenta, 5.8 pg/g TEQ for cord blood, 10.0 pg/g TEQ for postpartum blood and 10.2 pg/g TEQ for milk.