Passively transmitted gp41 antibodies in babies born from HIV-1 subtype C-seropositive women: correlation between fine specificity and protection.

HIV-exposed, uninfected (EUN) babies born to HIV-infected mothers are examples of natural resistance to HIV infection. In this study, we evaluated the titer and neutralizing potential of gp41-specific maternal antibodies and their correlation with HIV transmission in HIV-infected mother-child pairs. Specific gp41-binding and -neutralizing antibodies were determined in a cohort of 74 first-time mother-child pairs, of whom 40 mothers were infected with HIV subtype C. Within the infected mother cohort, 16 babies were born infected and 24 were PCR negative and uninfected at birth (i.e., exposed but uninfected). Thirty-four HIV-uninfected and HIV-unexposed mother-child pairs were included as controls. All HIV-positive mothers and their newborns showed high IgG titers to linear epitopes within the HR1 region and to the membrane-proximal (MPER) domain of gp41; most sera also recognized the disulfide loop immunodominant epitope (IDE). Antibody titers to the gp41 epitopes were significantly lower in nontransmitting mothers (P < 0.01) and in the EUN babies (P < 0.005) than in HIV-positive mother-child pairs. Three domains of gp41, HR1, IDE, and MPER, elicited antibodies that were effectively transmitted to EUN babies. Moreover, in EUN babies, epitopes overlapping the 2F5 epitope (ELDKWAS), but not the 4E10 epitope, were neutralization targets in two out of four viruses tested. Our findings highlight important epitopes in gp41 that appear to be associated with exposure without infection and would be important to consider for vaccine design.

In vivo anti-inflammatory effect of statins is mediated by nonsterol mevalonate products.

This study set out to clarify whether the inhibition of sterol or nonsterol derivatives arising from mevalonate biotransformation plays a major role in the in vivo anti-inflammatory action of statins. Hepatic synthesis of all these derivatives was inhibited in mice by administered statins, whereas squalestatin inhibited only sterol derivatives. Using a short-term treatment schedule, we found that statins reduced the hepatic activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase without affecting blood cholesterol. This treatment inhibited lipopolysaccharide- and carrageenan-induced pouch leukocyte recruitment and the exudate production of interleukin-6, monocyte chemotactic protein-1, and RANTES. Coadministration of mevalonate reversed the effect of statin on leukocyte recruitment. The inhibition of sterol synthesis by squalestatin did not have any anti-inflammatory effect, indicating that the biosynthesis of nonsterol compounds arising from mevalonate is crucial for the in vivo regulation of cytokine and chemokine production by statins. Their inhibition by statins may account for the reported anti-inflammatory effects of these drugs and may provide a biochemical basis for the recently reported effects of statins in the prevention of cardiovascular disease and mortality.

Oleamide-mediated sleep induction does not depend on perturbation of membrane homeoviscosity.

To verify whether the sleep-inducing properties of oleamide were related to its ability to perturb membrane homeoviscosity, affecting 5-HT(2A) receptors, we compared the effects of oleamide and oleic acid, the latter lacking both the sleep-inducing effect and the action on 5-HT(2A) receptors. In binding studies the two compounds did not directly interact with rat brain cortex 5-HT(2A) receptors, nor did they increase the affinity of a 5-HT(2A) agonist, either in vitro or ex vivo. They had similar fluidizing effects, in vitro at high concentrations (>/=10 microM), and ex vivo after a dose of 100 mg/kg, and they reduced locomotor activity with similar potency. There thus appears to be no causal relationship between the fluidizing effects of oleamide and its sleep-inducing properties.

Modulation of ATPase activity by cholesterol and synthetic ether lipids in leukemic cells.

Synthetic ether lipids (EL) exert their antiproliferative action on leukemic cells through localization in the plasma membrane with subsequent biochemical effects which are still being elucidated. In the present study, the modulation of membrane-linked ATPase activity was investigated in relation to changes in membrane fluidity of HL60 and K562 human leukemic cells. Incubation of HL60 and K562 cells with EL under non-cytotoxic conditions caused significant membrane fluidization which was related to the membrane cholesterol (CHOL) levels. HL60 cells, which are sensitive to the cytotoxic action of EL, had a lower basal CHOL content. When HL60 cells were loaded with CHOL, Na+, K(+)-ATPase activity was reduced significantly compared to that of untreated cells. In contrast, CHOL-deprived K562 cells had twice the Na+,K(+)-ATPase activity of unmodified K562 cells. Na+K(+)- and Mg(2+)-ATPase activities were stimulated significantly in both cell lines by EL at concentrations lower than 20 microM. This stimulation was greater in cells richer in CHOL, such as K562 cells and CHOL-enriched HL60 cells. In contrast, Na+,K(+)-ATPase in both cell lines was inhibited by EL above 20 microM regardless of the CHOL content. Mg(2+)-ATPase activity was not related to cell CHOL content and was not inhibited by EL above 20 microM.

Effect of paf antagonists on the cytotoxic activity of antineoplastic ether phospholipids.

The capability of the methoxy-substituted 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3 or Edelfosine) and the two aza-alkylphospholipids BN 52205 and BN 52211 to bind to the PAF receptor was analysed in rabbit platelet membranes. Ether phospholipid concentrations were tested between 10(-5) M and 10(-11) M. The results indicate that ether phospholipids are not able to bind to the PAF receptor and do not prevent PAF binding to its receptor. Moreover, the cytotoxic effect of three potent PAF antagonists, BN 52021, BN 50730 and BN 50739, were analysed in HL60 promyelocytic cells. These cells were pre-and co-treated with PAF antagonists and ether phospholipids. The data show that the three PAF antagonists failed to counteract the activity of ET-18-OCH3, BN 52205 and BN 52211 thus demonstrating that the cytotoxic effect of these new anti-neoplastic drugs is not mediated by the PAF receptor.

Membrane cholesterol content and sensitivity of human carcinoma-cells to antineoplastic ether phospholipids.

Ether phospholipids represent a new class of anti-cancer drugs which appear to exert their tumoricidal activity through a direct and indirect cytotoxic effect against tumor cells of different origins. The chemotherapeutic interest in these new drugs is based on the finding that, contrary to the majority of anti-cancer drugs, ether phospholipids do not interfere with DNA synthesis, are anti-invasive and induce tumor cell differentiation. There is increasing experimental evidence that the direct cytotoxic effect of these new drugs is mediated by the cell membrane. We have measured the lipid membrane composition of three human carcinoma cell lines that have been found to possess different sensitivity to the tumoricidal activity of four antitumor ether phospholipids. A statistically significant difference has been found in the membrane cholesterol content of the three cell lines and a positive correlation has been established between the membrane cholesterol level and the carcinoma cell sensitivity to ether phospholipids. These findings emphasize previous data obtained with leukemic cells and reinforce the interest in ether phospholipids whose cytotoxic properties may represent a new step towards a more promising anti-cancer chemotherapy.

Hypericum perforatum L. extract does not inhibit 5-HT transporter in rat brain cortex.

The hydroalcoholic extract of Hypericum perforatum L. is an effective antidepressant, although its mechanism of action is still unknown. It inhibits the synaptosomal uptake of serotonin (5-HT), dopamine and noradrenaline, suggesting a biochemical mechanism similar to the synthetic standard antidepressants. In the present study, further investigating this hypothesis, we confirmed that a hydromethanolic extract of H. perforatum inhibited [3H]5-HT accumulation in rat brain cortical synaptosomes with an IC50 value of 7.9 microg/ml. The IC50 of pure hyperforin was 1.8 microg/ml, so the activity of the total extract is not related only to its hyperforin content (<5%). This inhibitory effect, however, is not due to a direct interaction with, and blockade of, the 5-HT transporters since the extract, like hyperforin, did not inhibit [3H]citalopram binding (IC50 > 100 microg/ml and 10 microg/ml, respectively). We also found that 3-10 microg/ml of the extract, or 0.3-1 microg/ml hyperforin, induced marked tritium release from superfused synaptosomes previously loaded with [3H]5-HT. The releasing effect of the extract resembles the releasing effect of a reserpine-like compound (Ro 04-1284), i.e. it was slightly delayed and was 5-HT carrier- and calcium-independent. These data suggest that the hydromethanolic extract of H. peforatum, similarly to Ro 04-1284, rapidly depletes storage vesicles, raising the cytoplasmic concentration of 5-HT, and this increase is presumably responsible for the apparent inhibition of [3H]5-HT uptake. Therefore, our in vitro data do not confirm that the hydromethanolic extract of H. perforatum acts as a classical 5-HT uptake inhibitor but indicate reserpine-like properties. However, the concentrations of the active component(s) effective in vitro as reserpine-like agent(s) (i.e. corresponding to > or =3 microg/ml of the hydromethanolic extract) do not seem to be achieved in the brain after pharmacologically effective doses of the extract, as indicated by the finding that there were no significant changes of rat brain 5-HT and 5-hydroxyindoleacetic acid levels after a schedule of treatment (3 x 300 mg/kgday, orally) active in an animal model predictive of antidepressant-like activity. These data also suggest that the antidepressant effect of H. perforatum extracts is unlikely to be associated with interaction with GABA, benzodiazepine and 5-HT1 receptors since, in receptor binding studies, we found IC50 values higher than 5 microg/ml. Therefore other, still unknown, mechanisms are possibly involved in H. perforatum antidepressant effects.

A rapid electrochemical assay of lecithin in amniotic fluid using a fluoride ion-sensitive electrode.

An electrochemical method is described for the determination of lecithin in rat and human amniotic fluid. Choline is released from lecithin enzymatically by phospholipase D and the hydrogen peroxide released by the action of choline oxidase is quantitatively determined by peroxidase-catalyzed rupture of the covalent C-F bond of 4-fluorophenol. The concentration of F- ions in solutions is determined by a fluoride sensitive electrode from the resulting cell potential difference recorded before and 10 min after addition of a solution containing phospholipase D, choline oxidase and horseradish peroxidase. Lecithin levels in rat amniotic fluid increased from about 10 mumol/l on the 20th day of gestation to 80 mumol/l on day 21, which corresponds to the time of spontaneous delivery. In human amniotic fluid the lecithin concentrations determined with this new method parallel those already reported. They were approximately 10 to 50 mumol/l between the 15th and 18th weeks of gestation and increased from 5- to 7-fold between the 37th and 41st weeks of pregnancy. This method was only slightly influenced by the presence of blood or meconium contamination in the amniotic fluid.

Endotoxin regulates the maturation of sterol regulatory element binding protein-1 through the induction of cytokines.

Endotoxin (LPS), by raising the levels of cytokines, markedly influences lipid metabolism. To clarify the molecular mechanism of this effect, we examined the action of endotoxin in vitro and in vivo on the regulation of sterol regulatory element binding protein-1 (SREBP-1). In HepG2 cells stimulated with LPS, a dose-dependent increase in the level of the mature form of SREBP-1 was observed. For in vivo studies, endotoxin was administered intraperitoneally to CD1 mice fed with a standard or a cholesterol-enriched diet to increase the basal levels of circulating and liver cholesterol. Endotoxin raised cholesterol levels and stimulated the maturation of hepatic SREBP-1 in both normal and cholesterol-fed mice, indicating that the lipogenic effect of LPS was independent of endogenous sterol levels. To assess whether the lipogenic effect of endotoxin was linked to cytokine production, we administered LPS to C57Bl/6J endotoxin-sensitive and to C3H/HeJ endotoxin-resistant mice, which do not produce tumor necrosis factor in response to LPS. Significant induction of cholesterol levels and SREBP-1 activation was observed only in C57Bl/6J mice, indicating that cytokine production is crucial for the regulation of SREBP-1, and that the transcriptional activation of cholesterol biosynthesis may be part of the acute-phase response.

Protein kinase C is not involved in cholesterol-induced resistance to synthetic ether lipids.

The possible role of protein kinase C in cholesterol-induced resistance to ether lipids was investigated. The enrichment of HL60 cells in cholesterol (CHOL) (HL60-CHOL) resulted in a significant increase in the ID50 values for 1-octadecyl-2-methyl-rac-glycero- 3-phosphocholine (ET-18-OMe) (3.75 +/- 0.7 microM and 6.69 +/- 0.5 microM for HL60 and HL60-CHOL, respectively). In the same conditions, HL60 and HL60-CHOL cells showed comparable levels of both cytosolic and membrane-associated protein kinase C activity. Phorbol ester (PMA) stimulation induced protein kinase C to translocate from the cytosol to the plasma membrane in both cell types and with similar kinetics (272 +/- 32% and 299 +/- 41% increase in HL60 and HL60-CHOL, respectively after 100 ng/ml PMA for 10 min). Pretreatment of the two cell types with 50 microM ET-18-OMe resulted in comparable levels of PKC inhibition after phorbol ester stimulation. These results suggested that alterations in plasma membrane lipid composition induced by CHOL do not result in major changes in protein kinase C activity. Thus, protein kinase C does not appear to be involved in cholesterol-induced resistant phenotype in HL60 cells.

Liver DNA alkylation after a single carcinogenic dose of dimethylnitrosamine to newborn and adult CFW Swiss mice.

N-nitrosodimethylamine N-demethylase activity, DNA alkylation, capacity for O6-methylguanine repair and cell proliferation were measured in livers of newborn and adult CFW mice after a single carcinogenic dose of DMNA. DNA alkylation was found in newborn and adult mouse livers but it was significantly higher in the newborn. 6- and 7-methyl substitutions of guanine were identified by HPLC analysis in newborn and in adult mouse livers. Metabolic 14C incorporation into adenine and guanine was observed only in liver DNA of newborns. O6-methylguanine levels were higher in newborn than adult mice after a single i.p. dose of [14C]DNMA. Liver DNA repair capacity measured as O6-meG-DNA methyltransferase was higher in adults than in newborns. De novo liver DNA synthesis was more inhibited by DMNA pretreatment in newborn than in adult mice. The relationship between these parameters and the greater neonatal liver tumor susceptibility is discussed.

Aspartame and the rat brain monoaminergic system.

A high dose of aspartame (APM) was administered to rats to study possible effects on brain monoaminergic systems. APM and its metabolite phenylalanine (Phe) were given orally at doses of 1000 and 500 mg/kg, respectively. Significant increases were seen in brain Phe and tyrosine (Tyr) levels. Two different approaches were used to study monoaminergic systems: whole tissue measurements by HPLC-ED and in vivo voltammetry in freely moving rats. Dopamine, serotonin and their metabolites were taken as indexes of neuronal activity. In spite of the high dose used, no modification was found in monoamines or their metabolites in striatum, hippocampus and nucleus accumbens.

Potentials of liposomes in diagnosis and treatment of pulmonary metastases: an experimental study in the rat.

OBJECTIVE: The ultimate goal of the therapy of lung metastases is to destroy all malignant cells while sparing normal ones. Liposomes represent a novel approach for the selective transport of tracers and therapeutic agents to cancer cells because of their flexibility, low toxicity, wide range of possible variants, simplicity to make, and because agents can be entrapped in them in their native states in large amounts. We have studied the biodistribution of "Stealth" liposomes in the experimental model of lung metastases in the rat. METHODS: The secondaries were induced by i.v. injection 20. 10(6) cancer cells (DHD/K12/TRb line) in BD-IX rats. The study of the liposome biodistribution in the rat was carried out by the use of unilamellar liposomes with homogeneous size distribution (0.1 microns), the liposomes were labeled with Cholesteryl-Bodipy. The rats were sacrificed at scheduled times after the injection; blood, urine, metastatic and healthy lung, colon, liver and spleen were analysed by a microcytofluorimetric examination. RESULTS: Liposomes prolonged the circulation time of Cholesteryl-Bodipy. Only spleen and lung metastases exhibited an accretion of fluorescent liposomes. CONCLUSIONS: The biodistribution of such formulation of liposomes in rats with lung metastases, may be of considerable importance in diagnosis and therapy of the secondaries, for increasing the concentration of tracers and therapeutic agents in tumor tissue while minimizing the likelihood of aspecific distribution and toxicity to non target tissue.

Interspecies and interstrain studies on the increased susceptibility to metrazol-induced convulsions in animals given aspartame.

The ability of aspartame (APM) to increase the susceptibility to metrazol-induced convulsions was studied in two strains of mice (CD1 and DBA/2J) and in guinea-pigs. Rats were included as known positive controls. Plasma and brain levels of phenylalanine (Phe) and tyrosine (Tyr) were measured in CD1 mice and guinea-pigs at various intervals after a dose of 1 g APM/kg body weight (administered orally to mice and ip to guinea-pigs). In mice, peak levels of Phe and Tyr were observed in plasma after 30 min and in brain after 60 min. In guinea-pigs peak plasma levels of Phe and Tyr occurred 30 min after treatment. Phe was at a maximum in guinea-pig brain after 30 min, while Tyr levels reached a peak at 120 min. In further experiments Phe and Tyr levels were measured 1 hr after APM doses of 0.5, 0.75 or 1 g/kg. In CD1 mice, plasma Phe and Tyr levels were increased significantly only at the highest dose, whereas in brain, Tyr concentrations were significantly increased by 0.75 or 1 g APM/kg and Phe was significantly increased by all three doses. In the guinea-pig, plasma Phe and Tyr were increased significantly only by 1 g APM/kg and in brain this dose significantly raised only the Phe levels. Monoamine and metabolite levels were determined in the brain striata of CD1 and DBA/2J mice 1 hr after the oral administration of 1 or 2 g APM/kg body weight; no differences from control values were found in either strain. The studies of potentiation of metrazol-induced convulsions showed that APM, at doses of up to 2 g/kg body weight, had no such effect in mice or guinea-pigs. In contrast, as expected, the potentiation was significant in the rat at 1 g/kg.

Effect of tyrosine on the potentiation by aspartame and phenylalanine of metrazol-induced convulsions in rats.

Male rats were treated by oral intubation with tyrosine (Tyr), at doses of 0.5 and 1.0 g/kg body weight, alone or together with 1 g aspartame (APM)/kg body weight, or an equivalent dose of phenylalanine (Phe; 0.5 g/kg body weight); the effects on seizures induced by an effective dose of metrazol (ED50) were observed. Tyr (0.5 g/kg body weight) had a protective effect against the Phe-potentiation of metrazol-induced clonic-tonic convulsions. At the same dose Tyr had no effect on the seizure-promoting activity of APM, but at 1 g/kg it reduced the proconvulsant potential of the sweetener. Analysis of the brain and plasma amino acid concentrations indicated that the Tyr to Phe ratio tended to be enhanced in Tyr-Phe treated rats compared with those treated with Phe alone. This ratio remained essentially constant in the brain of APM-treated rats, compared with those treated with APM plus 1 g Tyr/kg body weight, whereas an increase in this ratio in the plasma was observed. These results confirm that Tyr antagonizes the proconvulsant effect of Phe and APM and they further suggest that no simple relationship exists between the relative brain concentrations of the two amino acids and the response to metrazol convulsions.

Plasma and brain kinetics of large neutral amino acids and of striatum monoamines in rats given aspartame.

Two doses (250 and 1000 mg/kg body weight) of aspartame were administered orally to male rats, and plasma and brain phenylalanine and tyrosine kinetic profiles were studied. In both plasma and brain the maximum increase in phenylalanine and tyrosine levels was reached 60 min after treatment. The changes in brain levels of phenylalanine or tyrosine 0, 60, 120 or 180 min after treatment with 1000 mg AMP/kg were directly correlated with the ratio of the plasma concentration of phenylalanine or tyrosine to the overall plasma concentration of the other large neutral amino acids. The time course of monoamine and metabolite concentrations, in the corpora striatum of the brain, was studied after an oral dose of 500 mg phenylalanine/kg. No significant modifications of monoamine levels were found at any of the times studied, up to 5 hr after dosing.

A novel pharmacological approach for paraquat poisoning in rat and A549 cell line using ambroxol, a lung surfactant synthesis inducer.

Paraquat (PQ) is a widely used herbicide that causes acute adult respiratory distress syndrome (ARDS) and chronic lung damage (diffuse fibrosis). One of the earliest biochemical effects induced by PQ is damage to type II pneumocytes with consequent depletion of surfactant. With the aim of counteracting the toxic effects of PQ, a series of investigations were performed into the possible protective effect of the drug ambroxol, which induces the synthesis of surfactant in lung alveolar type II cells. The number of survivors and survival time of rats treated ip with 35 mg PQ/kg was significantly increased by 3 days of ambroxol pretreatment and by ambroxol treatment 30 min or 2 hr after PQ. Total phospholipid content in lung and bronchoalveolar lavage fluid (BALF) was significantly reduced 30 hr after treatment with PQ alone. The association of ambroxol with PQ significantly antagonized this reduction. In BALF the ratio between palmitic acid and stearic acid concentrations was significantly lower in animals treated with PQ alone but was returned to normal by the association with ambroxol. The cell line A549, exposed in vitro to PQ concentrations from 0.5 x 10(-4) to 2 x 10(-3) M, showed a significant dose-dependent loss of viability. Cells pretreated with ambroxol (10 mg/ml) were more resistant to PQ and their viability started to decrease significantly only from a PQ concentration of 0.8 x 10(-3) M. Membrane microviscosity was measured on the same cells. Cells treated with PQ alone showed a reduction of membrane microviscosity, which was significantly counteracted by ambroxol pretreatment. The curves of modification of membrane microviscosity of cells treated with PQ and with ambroxol plus PQ paralleled those of cell viability, indicating that the stimulation of surfactant synthesis in vitro may be a prerequisite for counteracting some of the early effects of PQ.

The effect of culture medium composition on ether lipid cytotoxic activity.

The effect of a serum-free medium (TNB-100), compared to RPMI 1640 containing 10% fetal bovine serum (FBS), on the lipid composition of HL60 and K562 leukemic cells was investigated. The 10% FBS RPMI medium contained approximately three times more phospholipids (PL), about three times more protein and eight times more cholesterol (CHOL) than did the TNB-100 medium. Cells cultured in TNB-100 medium, referred to as HL60-TNB and K562-TNB cells, were significantly lower in PL and CHOL than 10% FBS RPMI cells, with about a threefold higher PL-to-CHOL ratio; however, these cells were significantly higher in protein content. Cells grown in TNB-100 were also significantly more fluid than 10% FBS RPMI cells and were more sensitive to the fluidizing action of the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine. The 50% inhibitory dose of the drug was about 50% lower in TNB-grown cells than in 10% FBS RPMI cells.

Alpha 1-antitrypsin variants produced by recombinant DNA: differences in elastase inhibitory activity and resistance to oxidant agents.

Inherited or "acquired" deficiency of alpha 1-antitrypsin (believed to be the cause of pulmonary emphysema) will probably be treated in the future by replacement with alpha 1-antitrypsin purified from human plasma or produced by recombinant DNA, which seems promising because it permits site-specific mutagenesis in the oxidizable active site of the normal human alpha 1-antitrypsin. The aim of this in-vitro study was to investigate the elastase inhibitory activity and the resistance to oxidizing agents of normal human alpha 1-antitrypsin, a recombinant yeast-produced variant (VAL 358) and a recombinant E. coli-produced variant (LEU 358). The inhibitors were exposed to chemical oxidants (NCS, H2O2, xanthine/xanthine oxidase, chloramine-T) and to PMA-activated neutrophils. The elastase inhibitory activity was assayed on porcine pancreatic elastase and neutrophil elastase. Normal alpha 1-antitrypsin and VAL 358 variant were good inhibitors of both elastases. LEU 358 variant was the best inhibitor for neutrophil elastase, but it poorly inhibited the porcine pancreatic elastase. Normal alpha 1-antitrypsin was affected by all oxidants; both variants were almost totally resistant to chemical oxidants and to activated neutrophils. We conclude that recombinant alpha 1-antitrypsin variants differ in their elastase inhibitory activity and offer increased resistance to oxidant agents.

Analytical validity of electrochemical determination of lecithin for establishing foetal lung maturity in normal and complicated pregnancies.

An electrochemical assay of lecithin for the prediction of foetal lung maturity in normal and complicated pregnancies has been analytically evaluated. The method is based on sequential enzymatic reactions causing the stoichiometric transformation of lecithin to hydrogen peroxide, which reacts with an organo-fluoro compound in the presence of peroxidase. The rupture of the C-F bond releases fluoride ions, that are detected by a selective electrode. The correlation between the lecithin concentration in amniotic fluid, measured electrochemically, and the fluorescence polarization (FP) value, chosen as reference method, was determined. Correlation studies were performed on rat amniotic fluids, on 67 samples from human normal pregnancies, and on seven samples from complicated pregnancies. The relationships between the FP value and the lecithin concentration were linear, and the correlation coefficients were 0.987 for rat and 0.884 for human amniotic fluids. Concordance was good for predicting foetal lung maturity in complicated pregnancies.