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PubMed1991

Isolation from human placental extracts of a preparation possessing 'early pregnancy factor' activity and identification of the polypeptide components.

Protocols are described for the isolation from human placental extracts of a preparation which is active in the rosette inhibition assay by inducing an increased rosette inhibition titre. In this respect the preparation mimics the effects of pregnancy sera, an ability ascribed to the presence in these sera of a so-called 'early pregnancy factor' (EPF). In addition to this activity in the rosette inhibition assay, the preparation was also shown to modulate the expression of cell surface immunoglobulin on peripheral blood mononuclear cells. The polypeptide composition of the preparation was relatively simple, as revealed by SDS-PAGE. There was a major 12 kd polypeptide previously isolated in a rosette inhibition titre-active preparation from ovine placental extracts, and a small amount of 68 kd polypeptide. N-Terminal amino acid sequence analyses obtained after blotting onto polyvinylidene difluoride membranes identified the latter polypeptide as serum albumin and the major 12 kd polypeptide as human thioredoxin. Gel permeation analysis partially resolved activity expression away from the 12 kd polypeptide as activity expression was also found to be associated with low mol. wt material. It is concluded that EPF activity expression in pregnancy sera involves molecules related to and associated with thioredoxin.

Amino Acid Sequence↗
PubMed1998

Human placental extract stimulates liver regeneration in rats.

The effect of human placental extract (HPE) on liver regeneration in rats was investigated. After intravenous administration of HPE to a-naphthylisothiocyanate (ANIT)-intoxicated rats, the labeling index in hepatocytes was significantly increased to a level 16.5 times higher than that of the control. A 1/500 dilution of HPE directly stimulated DNA synthesis of the hepatocytes in primary culture. HPE heated at 121 degrees C did not stimulate the labeling index in vivo or hepatocyte DNA synthesis in primary culture, suggesting that HPE contains heat-unstable but potent mitogens for hepatocytes. HPE contains hepatocyte growth factor (HGF), but the mitogenic effect of HPE cannot be explained by the effect exerted by HGF alone, since both the labeling index in vivo and hepatocellular DNA synthesis in vitro stimulated by HPE were much higher than those stimulated by HGF alone when the applied doses of HGF were set to be almost the same level between each case. When HPE was fractionated on a heparin-sepharose column, the mitogenic effect of HPE was found to be located mainly in the heparin-bound fraction. Hepatocyte DNA synthesis induced by this fraction was enhanced cooperatively by the heparin-unbound fraction, suggesting that there are some modulators in the heparin-unbound fraction which enhance the proliferative activity of the heparin-bound fraction by a synergetic mechanism. Both HPE and heated HPE completely recovered the biochemical marker activity for liver function (glutamic-pyruvic transaminase, GPT; alkaline phosphatase, ALP; lactate dehydrogenase, LAP; gamma-glutamyltransferase, gamma-GTP activities and the bilirubin concentration) almost to the control level in the serum of ANIT-intoxicated rats, indicating that HPE also contains a heat-stable fraction which repairs liver function.

Animals↗
PubMed1986

Maternal alloimmune reactions towards the murine conceptus and graft-versus-host reaction (GVHR). II. Inhibition of priming by placental extracts.

Gestation can induce a priming for a GVHR towards paternal strain antigens, although this priming is significantly lower than the one induced by experimental immunization. A role has been sought for placental substances in decreasing this priming through immunomodulation. BALB/c (H-2d) spleen cells do not usually induce a systemic, lethal GVHR in DBA/2 (H-2d) newborn mice except when the donors are preimmunized with DBA/2 cells. Placental extracts (as well as RPMI medium or liver extracts used as controls) were added to DBA/2 cells injected into BALB/c mice used as cell donors for GVH induction. The latter's spleen cells, harvested on day 6 after immunization, were used for systemic and local GVHR. In the systemic assay (lethal effect on DBA/2 newborn mice injected i.v. with BALB/c spleen cells) a significant protection was observed. In the local assay (popliteal lymph node assay in F1 hybrids injected with BALB/c spleen cells into the foot-pad) a highly significant inhibition of priming was detected in recipients injected with spleen cells from placental extract-treated donors. The stimulation index was even lower than that obtained with unprimed BALB/c spleen cells. The same type of local GVHR in (CBA/Ca X A/J) F1 hybrids injected with CBA cells led to similar results. In both situations (systemic and local GVHR) the observed inhibition was found to be specific to the priming cell strain. These results support the working hypothesis that placental substances are able to modify the systemic response of an organism towards both H-2 and non-H-2 alloantigens.

Animals↗
PubMed1983

Double-stranded RNA-stimulated enzyme activities isolated from human placental extracts.

A (2'-5')An synthetase activity was isolated from human placental extracts by affinity chromatography on poly(rI) . poly(rC)-agarose. The oligonucleotide (2'-5')An was identified by (1) chromatography on PEI-cellulose and DEAE-cellulose, (2) inhibition of polypeptide synthesis in lysed rabbit reticulocytes (3) competition of the binding of pppA(pA)3,3'-[32P]pCp to rabbit reticulocyte lysates, and (4) alkaline phosphatase digestion. The synthetase activity in most placental preparations is activated by natural or synthetic dsRNA. However, in a few placental synthetase preparations, dsRNA is only marginally stimulatory and only becomes effective by prior treatment of the enzyme preparations with the calcium-dependent micrococcal nuclease. This suggests that there is an endogenous placental dsRNA contaminant in the enzyme preparations. In some synthetase preparations, a second dsRNA-stimulated product, tentatively identified as the nucleotide 5'-IMP, is also observed. Because the specific AMP deaminase inhibitor coformycin (10 microM) blocks the formation of IMP from ATP and causes a quantitative accumulation of AMP, and because the formation of IMp becomes independent of dsRNA when ADP or AMP is used in place of ATP, the presence of a dsRNA-stimulated ATP phosphohydrolase (ATPase) activity in human placenta is suggested.

2',5'-Oligoadenylate Synthetase↗
PubMed2002

A human placental extract: in vivo and in vitro assessments of its melanocyte growth and pigment-inducing activities.

BACKGROUND: The authenticity of various prototype human placental extracts with biological activity, such as that inducing vitiligo repigmentation, is under serious criticism, mainly due to a lack of demonstration at the cellular level. Considering the present worldwide scenario with regard to the occurrence and treatment of vitiligo, a thorough scientific exploration of such extracts should be undertaken. METHOD: One such prototype placental preparation was prepared, and was evaluated with regard to its melanogenic action in C57BL/6J mice in vivo and its mitogenic and melanogenic activity on B16F10 mouse melanoma cells and normal human melanocytes in vitro. The extract was applied topically to mice with age-induced prolonged telogenic phase of hair growth (grey body coat hair). Standard 3H-thymidine incorporation and spectrophotometric methods were followed to illustrate mitogenic and melanogenic effects at the cellular level. RESULTS: The resurgence of blue skin, followed by shiny black hair, at the regions of application of the extract demonstrated the reversal of the age-induced prolonged telogenic phase of hair growth to the anagenic phase after topical application of the extract on C57BL/6J mice. Further support was obtained from histology where, at the extract-treated sites, the development of new melanogenic centers and hair follicles was observed. During in vitro studies, the vehicle-free extract constituents stimulated both mitogenesis and melanogenesis of B16F10 mouse melanoma cells in a concentration-dependent manner. The cell morphology and extent of melanogenesis also showed significant changes. In addition, two known melanocyte activity-modulating peptides, endothelin-1 (ET-1) and adrenocorticotropic hormone (ACTH), were determined in the extract, chiefly in the total lipid fraction, indicating their effective cutaneous permeation. CONCLUSIONS: The extract was found to be a potent mitogen in the in vitro condition and a potent melanogen in both the in vitro and in vivo situations. This strongly suggests its therapeutic potential for the repigmentation of vitiligo patches.

Animals↗
PubMed1981

An approach to chemical characterization of human placental extracts: proteins, peptides, and amino acids analyses.

A procedure is described for characterization of the proteinic, peptidic, and amino-acidic components of human placental extracts. Four different preparations were analyzed. The first was an extract prepared in our laboratory from fresh on-term placenta. The other three were commercial products obtained by different procedures including autolysis and sterilization. The patterns given by each type of preparation are reproducible and characteristic.

Amino Acids↗
PubMed1995

Hydroalcoholic human placental extract: skin pigmenting activity and gross chemical composition.

BACKGROUND: Vitiligo is a pigmentary disorder of the skin of unknown etiology. It is thought to be of autoimmune origin after demonstration of antibody-mediated destruction of melanocytes. Photochemotherapeutic PUVA therapy is widely used in vitiligo with about 33% success. Aqueous or hydroalcoholic extracts of human placenta of ill-defined composition have also been used therapeutically for vitiligo. A hydroalcoholic human placental extract has been developed by us with pigmenting activity based on experimental therapies. Its chemical analysis was the primary objective of this study. METHODS: For the guinea pig experiment, 20 drops of the extract or vehicle (60% alcohol) as control was topically applied around the nipples covering the areola zones of male immature white guinea pigs (wt. 175-250 g) daily for 60 days with 15 minutes infrared (IR) exposure used for vascular dilatation and enhancement of the absorption of the extract. Standard methods have been followed for all chemical analyses. RESULTS: The guinea pig experiment showed clear pigmentation and hypertrophy of the experimental nipples to varying degrees. Chemical analysis of the extract revealed the presence of small-molecular-weight proteins/peptides, lipids (including glycosphingolipids), carbohydrates, sialic acids, cholesterol, triglycerides, high density lipoproteins (HDL), and others, including amino acids, nucleotides, carotenes, vitamins, etc. CONCLUSION: Glycosphingolipids, known modulators of B and T cells, were reported capable of inducing adhesion, spreading, and motility of melanoma. It is present in the extract and, therefore, may lead to skin pigmentation through induction of melanocytes. Endothelin, a 21-amino acid peptide, detected in human placenta and possibly extractable by our process, has been reported to be indispensable for melanocyte growth.

Absorption↗
PubMed1991

Growth suppression of transformed cells by a human placental extract not related to transforming growth factor beta.

We have examined whether human placental extracts contain tumour-growth-inhibitory factors. One fraction (EAP) from such extracts inhibited growth, in soft agar, of Ha-ras-transformed BALB/c 3T3 cells and human squamous lung carcinoma A-2182 cells. However, this fraction had no effect on the anchorage-dependent growth of these cells, although there was a slight mitogenic activity on nontransformed cells. These data together with those on plating efficiency indicated no significant cytotoxicity of EAP on transformed cell lines. Although this fraction contained transforming growth factor beta (TGF beta), this cannot account for its inhibitory activity, since (a) pure TGF beta does not inhibit anchorage-dependent growth of Ha-ras-transformed BALB/c 3T3 cells, (b) EAP retains its inhibitory activity in the presence of antibodies against TGF beta and (c) the inhibitory activity did not copurify with TGF beta. Partial characterization of our inhibitory factor suggests that the inhibitory factor is a new tumour-growth-inhibitory factor.

Animals↗
PubMedDate not supplied

Effects of human placental extract on hepatic drug metabolizing enzyme.

Effects of acute or subchronic administration of human placental extract (HPE), a worldwide clinically used agent, on hepatic drug metabolizing enzyme activities were evaluated in rats. Hepatic microsomal cytochrome P-450 (Cyt. P450) and cytochrome b5 (Cyt. b5) contents and cytosolic glutathione S-transferase (GST) activities were maximally induced after various periods of time following a single intraperitoneal injection of HPE (4 ml/kg) whereas microsomal UDP-glucuronyltransferase (UDPGT) activities were inhibited significantly. All these altered effects were returned almost to the basal levels after 96 h of treatment. Subchronic treatment (30 days) with HPE (1,2 or 4 ml/kg) afforded a significant induction of Cyt. P-450 and Cyt. b5 levels and that of GST activities with a concurrent suppression of the activities of UDPGT and these results were found to be dose-dependent. However, microsomal NADPH cytochrome c reductase activity was not affected either by acute or subchronic treatment. The observed variations in the levels and activities of above house-keeping enzymes were discussed in relation to the possible carcinogenic risk of long-term treatment with this pharmaceutical agent.

Animals↗
PubMed1985

Deviation of humoral and cellular alloimmune reactions by placental extracts.

Modifications of the alloimmune response at both the humoral and the cellular levels by placental extracts (PE) syngeneic to the recipient were studied in the mouse using two different H-2 strain combinations. CBA (H-2k) or C57BL/Ks (H-2d), immunized with A/J (H-2a) spleen cells. The tests included in vivo tumor allograft evolution (accelerated rejection or enhancement reactions), and in vitro analysis of the involved immune agents, both cellular and humoral, using mixed lymphocyte reactions (MLR) and biological activity studies of serum samples. Animals from the recipient strains exhibited a delayed rejection of A/J tumor Sa 1 allografts if preimmunization was carried out with 10(6) A/J spleen cells combined with PE syngeneic to the recipients, as compared to controls immunized with A/J cells only or supplemented with isogeneic liver extracts (LE). The serological analysis revealed that PE treatment did not modify the overall hemagglutinating antibody production but resulted simultaneously in both a decreased production of cytotoxic complement fixing antibodies and an increase of specific anaphylactic mast cell degranulating antibodies, as compared to controls. The sera from PE-treated donors also demonstrated enhancing activity following passive transfer to isogeneic recipients. MLR regulatory activity was exhibited by spleen cells from PE- and immunogen-treated mice although the same or stronger activity was obtained from mice immunized without the addition of PE. However, in vivo transfer of these cells to syngeneic recipients showed that PE treatment erased the accelerated rejection caused by allogeneic immunization in the absence of PE and could even cause some degree of allografted tumor enhancement. The cells responsible for this inhibitory effect were mainly IJ+ lymphocytes, since their elimination with a relevant anti-IJ serum and complement restored a secondary type rejection pattern. These results show that PE present during the onset of immunization can promote the activation of regulatory agents such as enhancing antibodies and suppressor cells favoring allograft survival.

Animals↗
PubMedDate not supplied

Effects of placental extracts on the immune response to histocompatibility antigens: class deviation of alloantibody response and allograft enhancement.

Placental extracts obtained from CBA (H-2k) mice during syngeneic (CBA female X CBA male) pregnancy were evaluated for their capacity to deviate the immune reaction of CBA mice toward A/J(H-2a) immunizing spleen cells, as tested by alloantibody subclass formation and Sa 1 allograft accelerated rejection-or enhancement. The immunomodulatory activity appeared to be located in the soluble and in the insoluble extracts of placenta. The sodium deoxycholate (SDO) solubilized proteinic material, fractionated on Concanavalin A [Con A] and injected to CBA mice simultaneously with A/J spleen cells caused a suppression of the hemagglutinating titer and C-mediated cytotoxicity (IgG2). However, it also favoured antibody-mediated mast cell degranulation (IgG1) and Sa 1 allograft enhancement. The presence and role of IgG1 antibodies in both tumor enhancement and gestation has been described. It is concluded that placenta contains Con A-binding glycoproteins which are located in the membranes and released in soluble form. They exert their immunomodulatory effect in a way which might help the successful outcome of pregnancy as it helps to enhance the development of allografted sarcoma.

Animals↗
PubMed1985

Catecholamines in arterial and venous umbilical blood: placental extraction, correlation with fetal hypoxia, and transcutaneous partial oxygen tension.

In 34 parturient women the levels of free epinephrine (E), norepinephrine (NE), and dopamine (D) were determined by a radioenzymatic method using maternal venous and umbilical arterial and venous blood. The study was conducted to investigate the relationship between fetal catecholamines and hypoxia, fetal heart rate (FHR), and transcutaneous pO2 (tcpO2). The placental catecholamine extraction rates were also calculated. Results The NE concentrations (10,200 pg/ml) and the E concentrations (1,120 pg/ml) in the fetal arterial blood were highly elevated with mean values increased 4-fold over umbilical vein values. Compared with the maternal venous blood, NE values were increased 20-fold, and E values 10-fold. Free D concentrations in fetal arterial blood (130 pg/ml) had risen 2.5-fold over maternal levels. These results suggest that the catecholamines measured in cord blood are of fetal origin and that the placenta has a high capacity for inactivation of free catecholamines. The placental extraction rate is 77 +/- 14% for NE, 76 +/- 16% for E, and 33 +/- 25% for D. The placental extraction rates for E and NE were virtually identical; in agreement with morphological studies they demonstrated absence of sympathetic innervation on the fetal side of the placenta. Highly significant correlations were found between fetal arterial NE concentrations and the 1-minute APGAR score, pH and base deficit in the umbilical artery and alterations of the FHR (deceleration area, baseline FHR). Further analysis of FHR alterations reveals that an increase in deceleration area without tachycardia is not correlated with an increase of fetal arterial NE concentration. A significant rise in NE was only found with additional tachycardia which is often associated with a loss of oscillation amplitude. Fetal arterial E concentrations were found to correlate with the fetal parameters indicating increased adrenal secretion of the hormone during fetal stress. However, correlation coefficients were lower than those obtained for NE. A significant effect of fetal hypoxia on arterial and venous D levels could not be demonstrated. Fetal tcpO2 varies between 0-25 mm Hg during the last two hours before delivery. In most cases tcpO2 was lower than the arterial pO2. Besides epidermal thickness and artifacts, skin perfusion is a major factor influencing the tcpO2 (transcutaneous arterial pO2 difference). Vasoconstriction of the cutaneous vessels induced by increased NE secretion during hypoxia may obviously produce a fall in tcpO2.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed2004

Analysis of fluorescence excitation-emission matrix of multicomponent drugs. A case study with human placental extract used as wound healer.

The possibility of finger printing of multi component drugs, where a number of components are fluorescent, has been explored after generation of excitation-emission matrix (EEM) contour plots. With nicotinamide adenine dinucleotide, reduced form (NADH) and N-acetyl tryptophanamide (NATA) serving as reference compounds, the contour widths were found to be proportional to fluorophore concentration. EEM generated from different batches of processed human placental extract, used as wound healer, in presence and absence of extraneous fluorescence probes like 1-anilino 8-naphthalenesulphonic acid (1-ANS) or dansyl chloride were found to be highly consistent. Variation of contour widths of these EEM after excitation at 280 or 340 nm were essentially zero subject to the limit of detection of variation (n = 5). This high degree of consistency between batches has been supported from analysis with such independent techniques like UV-absorption spectra, Fourier Transformed IR spectra, TLC and reverse phase HPLC. The advantages of EEM analysis have been discussed.

Chromatography, High Pressure Liquid↗
PubMed1984

Endogenous immunoreactive digitalis-like substance in neonatal serum and placental extracts.

Therapeutic levels of digoxin in the serum of untreated neonates delivered to mothers who had not received the drug prenatally were detected by radio-immunoassay. Digoxin levels in neonates should be interpreted with care because of the unknown contribution by the endogenous digitalis-like substance (DLS) to the level of the drug. Three commercially available radio-immunoassay kits were compared with regard to their sensitivity and reproducibility in detecting the endogenous DLS. The kit from Clinical Assays (Cambridge, Mass., USA) was selected for further investigations. In a series of 35 paired samples of maternal and cord blood the average DLS values in terms of digoxin were 0,52 +/- 0,07 and 0,81 +/- 0,27 ng/ml respectively. This difference is statistically highly significant. In the case of infants with DLS values of 1-1,5 ng/ml in terms of digoxin, approximately 1 week was required to reach nontherapeutic digoxin levels, i.e. below 0,5 ng/ml. Gel chromatography showed that the DLS in neonatal serum was more closely associated with protein than is authentic digoxin. In placental extracts it followed the elution profile of the protein completely, but it shifted to fractions with a lower molecular weight than haemoglobin after trypsinization. The level of DLS in neonatal serum was also increased by more than half its original value by trypsinization. Proteolysis therefore seems to have a releasing effect on DLS. The molecular size of this substance is probably in the same range as that of polypeptides, since it was not dialysable from trypsinized and untreated samples through a membrane with a 22 000 dalton molecular weight cut-off point.

Adult↗
PubMedDate not supplied

The growth of malignant and nonmalignant human cells is modulated by a human placental extract.

BACKGROUND: In the present paper, malignant and nonmalignant human cells were compared in their response to a fraction (fraction D, FD) of a human placental extract. MATERIALS AND METHODS: The activity of FD was tested on cell proliferation both in the absence and in the presence of 5%, 10% and 15% fetal bovine serum (FBS). For cells growing in monolayers, the medium was renewed with fresh medium containing FD 24 hours after plating and 3 days after the first exposure. In breast cancer cells only, it was also changed after 6 days. For leukemic cells, which grow in suspension, FD was added directly to the medium the day of the seeding and then after 3 and 6 days. RESULTS: In normal fibroblasts, when plated at a low density, a strong inhibitory effect on cell growth was seen with the highest FD dose. This effect was observed in the presence of 5% and 10% FBS, while it disappeared with 15% FBS. In endothelial cells, FD, in the presence of 5% or 10% FBS, produced a modest but constant inhibition of cell proliferation, which was evident after a short treatment and with almost every dose of FD. Breast cancer and leukemic cell lines, plated at a standard density, were markedly inhibited by FD, but this effect was reversed in serum-free conditions, at least in mammary cells. In leukemic cells, after an initial stimulatory effect, FD was not able to counterbalance the absence of serum. CONCLUSIONS: Our data seem to suggest that in FD both stimulating and inhibitory growth-factors coexist, the activity of which are greatly influenced by the culture conditions used.

Cell Division↗
PubMedDate not supplied

Anti-inflammatory effect of human placental extract: a biochemical mechanistic approach.

Significant increase of liver succinic dehydrogenase (SHD) activity was produced by carrageenin-induced edema in rats. Pretreatment with human placental extract "Placentrex" inhibited the increased liver SHD activity in a dose-dependent manner. "Placentrex" was found to have no effect on the liver SHD activity in normal rats. Furthermore, heat-induced erythrocyte lysis was inhibited to a substantial extent by "Placentrex" and was found to be almost dose-responsive. However, adenosine diphosphate (ADP)-induced platelet aggregation and trypsin activity were not changed in vitro by the "Placentrex". No alkaline phosphatase activity was found in this preparation. All these studies indicate that the membrane stabilization and depletion of adenosine triphosphate (ATP) synthesis may be the basis of anti-inflammatory effect of this drug.

Animals↗