Junctions and adhesion molecules in first trimester and term human placentas.

Placental tight and gap junctions and their adhesion molecules were studied by immunochemistry and electron microscopy in early and term placentas in order to clarify their pattern of expression during placental development. Early syncytio-cytotrophoblast contained tight junctions with occludin and gap junctions with connexins 40 and 43. At term, endothelial cells from arterioles had tight and gap junctions following each other. Occludin, claudins 3 and 5 were found at the paracellular clefts of endothelial cells together with connexins 32, 40 and 50. Stromal cells had mixed tight and gap junctions with connexins 32, 43, 50. Capillaries demonstrated interendothelial tight junctions with claudins 3 and 5, and small gap junctions. Taken together these observations showed that the numerous tight and gap junctions of the early placental syncytio-cytotrophoblast are observed in the foetal arterioles and capillaries in the term placenta. We conclude that the tightness of the placenta due to the junctions lying in the syncytio-cytotrophoblast in early pregnancy is maintained by the foetal endothelial layer in term pregnancy, with significant developmental changes of their transmembrane proteins.

Immunocytological evidence for hematopoiesis in the early human placenta.

Hematopoiesis has previously been observed in the human yolk sac, in placental villi and in the embryonic aorta. Here, our immunocytological study at 24 and 35 days showed packed erythroblasts in the placental vessels, mitotic figures and anti-Ki-67 reactions within these cells. Morphologically, the erythroblasts and vessels were similar to those found in the yolk sac during primitive hematopoiesis. In addition, numerous extravascular erythroblasts were found in the villous core. Positive reactions were obtained in erythroblasts using antibodies against glycophorin-A, GATA-2 and C-kit that characterize the hematopoietic cells. However, erythroblasts did not react with anti-CD34 and anti-CD45. In this respect, they differ from the hematopoietic cell clusters observed in the aorta of the human embryo. The staining for glycophorin-A was maintained in erythroblasts at 6-7 weeks and 12-14 weeks. Anti-GATA-2 reaction was decreased in erythroblasts and appeared in the perivillous cytotrophoblast. Anti-C-kit signal was detected in endothelial cells at 6-7 weeks and switched to stromal and perivascular cells at 12-14 weeks. By term, anti-GATA-2 staining was still present in the trophoblast and appeared in vessels while anti-C-kit was negative. For the leukocytes marker CD15, a staining was found in the endothelium at 35 days, 6-7 and 12-14 weeks and in leukocytes at term. CD45 antibody decorated the leukocytes at 12-14 weeks and at term. Erythroblasts undergo a primitive hematopoiesis in the early placental vessels that may be of value for the embryo in a period of low oxygen environment.

Separation of trophoblastic and vascular cell lineages and vascular maturation in early human placental development.

The trophoblast and vascular cell lineages have been studied by immunohistochemistry at 6 and 12-14 weeks of pregnancy. Perivillous and extravillous cytotrophoblasts were specifically stained by anti-cytokeratin 7 whereas endothelial cells were labelled by anti-CD34 at these two stages of pregnancy. Perivillous and extravillous cytotrophoblasts together with erythroblasts showed mitotic figures and anti-Ki67 positive nuclei at the 6th week. In the perivillous cytotrophoblast, the number of Ki67 positive nuclei decreased by 12-14 weeks and the staining was limited to the proximal extravillous trophoblast of cell islands. Some endothelial and perivascular cells were labelled with anti-Ki67 at 12-14 weeks. Erythroblasts did not stain at all at this stage. Endothelial cells bound lectin UEA1 and vessels exhibited a fluorescent signal after anti-myosin staining at 12-14 weeks. These data showed that the cytotrophoblast and endothelial cell lineages are not related from 6 to 12-14 weeks of pregnancy. Because mitotic figures or anti-Ki67 staining were not observed in endothelium or perivascular cells at the 6th week, it seems likely that the endothelial cells committed to vasculogenesis derived from stromal cells. By 12-14 weeks, endothelial cells had nearly achieved their maturation by acquiring 1-fucosyl-binding sites revealed by UEA1-lectin binding and insuring their own renewal by mitosis. The maturation of perivascular cells at 12-14 weeks was shown by the anti-sm-myosin staining. We conclude that placental vasculogenesis involves mesenchymal cells rather than trophoblast. The differenciation of vessels organizing from random plexi to a vascular arborescence may involve paracrine regulatory loops with the trophoblast.

Placental leptin receptor isoforms in normal and pathological pregnancies.

Alternate mRNA splicing of human leptin receptor generates four membrane isoforms with different C-terminal sequences. They differ by the length of their intracellular domain which include specific motifs crucial for the specificity of leptin signalling. As a step towards functional studies, we have characterized leptin receptors in human placenta from normal pregnancies and pregnancies associated with diabetes and pre-eclampsia. Leptin and leptin receptors were visualized by immunohistochemistry of placentas obtained from first and third trimester pregnancies. Antibodies against N and C-terminal epitopes showed signals in the apical membrane of the syncytiotrophoblast in early and term placental villi as well as in JAr and BeWo derived trophoblast cells. In addition, a distinct isoform recognized by its extracellular juxtamembrane epitope was exclusively localized in cytotrophoblast cells and likely stains the soluble receptor. At contrast with the transmembrane receptors, the expression of this isoform is increased in placentas of pre-eclamptic and diabetic women which synthesize more leptin than placenta from uncomplicated pregnancy. These data demonstrate that short and long transmembrane leptin receptors are expressed in the trophoblast and indicate that leptin synthetized within the placenta can act locally through both receptor isoforms. Being also accessible to leptin from maternal origin, these transmembrane receptors may signal differently in pregnancy with normal and increased leptin production. The co-localization of leptin and the soluble receptor isoform suggests that this isoform serves for modulating maternal free leptin levels through modification of leptin binding capacities.

Characterization of first trimester human fetal placental vessels using immunocytochemical markers.

Cell differentiation markers on placental villi from the first trimester of human pregnancy have been studied by indirect immunofluorescence. Fluorescence labelling with antibodies against CD34 and CD31 was conspicuous in the vascular cells. The vascular paracellular clefts were labelled by anti-cadherin-5. A few vascular cells exhibited a positive reaction for von Willebrand factor, high-molecular-weight melanoma-associated-antibody and alpha-sm-actin compared to term pregnancy, indicating changes in protein expression during vascular differentiation. The poor anti-collagen IV reaction and the absence of a sm-myosin fluorescent signal observed around the vessels confirned the immaturity of the vessels. In contrast, strong reactions have previously been obtained with the latter antibodies in similar locations using term placental villi. A labelling was observed for antibodies against alpha3 and alpha5 integrins in these immature placental vessels suggesting cell-matrix interactions with specific domains of laminin or fibronectin. The vascular cells were also stained by anti-CD26. Surprisingly, the fetal vascular cells exhibited immunostainings in common with the villous cytotrophoblast (CD26) or the syncytiotrophoblast (cadherin-5) and cell islands cytotrophoblast (CD31, cadherin-5, alpha3 and alpha5 integrin subunits). These observations suggested a two step process for fetal vasculogenesis in the villi: i/ the formation of peripheral vessels induced by growth factors or cytokines derived from the nearby trophoblast, ii/ the development of muscular vessels due to growth factors or cytokines production induced by circulatory changes.

Pericytes of term human foeto-placental microvessels: ultrastructure and visualization.

Placental microvessels examined by transmission electron microscopy showed many pericytes and pericyte foot processes included in the basement membrane. The foot processes were distinct from the endothelial cell extensions because of their lighter staining, their content of reticulum endoplasmic, their scarcity of microfilaments and their separation from endothelial cells by basement membrane material. Some of these features were held in common with early villous perivascular cells and pericytes from other microvascular beds. Denudation of the villi by dissection allowed the microvascular basement membrane to be reached and pericytes to be identified by scanning electron microscopy as large cells with extensions apposed to microvessels. Similarly, a two-step collagenase-dispase digestion with an intermediate Percoll gradient separation progressively removed the trophoblast and the stroma down to the microvessels. Pericyte-like cells were observed lying over the microvessels that exhibited some terminal dilatations where endothelial cells were still surrounded by the basement membrane. The origin and the potential functions of the pericytes (contractility, basement membrane secretion, angiogenesis and phagocytosis) in the placental vascular bed are discussed.

Isolation of villous microvessels from the human placenta.

A method for isolating the microvessels of the human placental villi has been developed in order to culture perivascular cells. It consists of an initial selection of the villi by serial sieving. The villi retained by the 75 microns sieve were digested by collagenase-dispase. A Percoll gradient permitted the isolation of microvessels still surrounded by stromal fibres and cells. Another digestion by collagenase-dispase eliminated the contaminant elements and allowed, after a new Percoll gradient, microvessels with endothelium, basement membrane and a few perivascular cells to be obtained. Each step of the isolation of microvessels was monitored by light or electron microscopy. Our study confirms the isolation of microvessels embedded in their basement membrane and the preservation of endothelial and perivascular cells after digestion. This method, which has permitted the culture of placental endothelial cells and pericytes, appears of interest for studying microvascular angiogenesis and permeability.

Phenotype of cultured fetal perivascular cells from human placenta studied by scanning electron microscopy.

The phenotype of perivascular placental cells has previously been studied using tissue sections from the fetal villi. The examination of these cells in culture by scanning electron microscopy gives us the opportunity to observe their three-dimensional phenotypes and associations outside their normal constraints. Human umbilical endothelial cells, which have a phenotype comparable to that observed in other studies, seem more flattened in culture than in their usual environment. Microvascular endothelial cells did not attain an epithelioid phenotype with close contacts between cells but formed a network of branched, elongated cells with phagocytotic activity. Some circular associations were observed when using a gelatinized matrix. Microvascular pericytes were large, flattened cells with an irregular border that pushed up nodular associations on a gelatin matrix. Chorioplacental myocytes adopted a network template comparable to that developed by microvascular endothelial cells. However, these elongated cells were thicker, without microvilli, and superficial filaments could be observed. In culture, confluent endothelial cells from the umbilical cord or microvascular pericytes associated as nodules reached a cell phenotype close to their in vivo counter-parts. This attainment of an in vivo phenotype remains questionable for chorioplacental myocytes. Microvascular endothelial cells, however, though there was sparse formation of circular associations, remained far from their in vivo phenotype.

Culture of endothelial cells from human placental microvessels.

Endothelial cells are known to participate in angiogenesis, adaptation of vascular tonus and maintenance of blood fluidity in the microcirculation. To investigate these functions in the placenta, we devised a method of isolation and culture of endothelial cells from villous microvessels. In primary culture, these intraplacental endothelial cells exhibited many features observed in microvascular endothelium from other organs: spindle-shape, rosette associations, circular arrangements and confluence. In contrast to the confluent endothelial cells derived from the umbilical vein, cells from microvessels did not form a cobblestone network. After trypsin digestion of microvessels, magnetic microbeads coated with S-Endol immunoglobulin, antithrombomodulin and Ulex europaeus-I lectins were tested for sorting endothelial cells. Only the microbeads coated with antithrombomodulin allowed a suitable magnetic cell separation after trypsinization. By contrast, the microbeads coated with each of these antibodies or with lectins attached to confluent cells from the second passage. The microbeads detached from the cells at different rates. Their examination by scanning microscopy indicates that a portion of these microbeads was phagocytosed. Microvascular endothelial cells from the second passage were intensively stained by the anti-von Willebrand reaction and only weakly by the anti-smooth muscle alpha-actin reaction. They incorporated acetylated-low density lipoproteins coupled to a fluorescent probe. The positive reactions against the anti-von Willebrand factor and the uptake of the fluorescent acetylated-low density lipoproteins were modified after eight passages.

Pharmacokinetic parameters of antipyrine in dog after hepatectomy.

Pharmacokinetic studies with antipyrine were carried out on beagle dogs to determine the consequence of hepatectomy on hepatic drug metabolizing capacity, the rate of hepatic regeneration, and the possible beneficial effect of hepatocellular transplantation. The drug (250 mg) was administered by short IV infusion in three groups of dogs (first group, 65% hepatectomy; second group, 65% hepatectomy with hepatocyte transplantation; third group 80% hepatectomy). Pharmacokinetic parameters of antipyrine were evaluated before surgery and within 10 d after surgery. Blood samples were taken at frequent intervals after drug administration and antipyrine was assayed in plasma by a specific HPLC method with UV detection. Before surgery, the mean elimination half-life was about 1.1 h and total clearance averaged 6 L h-1. In dogs with 65% hepatectomy, no statistical differences in pharmacokinetic parameters of antipyrine appeared before or after surgery. When 65% hepatectomy was associated with hepatocyte transplantation, a significant increase in elimination half-life and a significant decrease in total clearance were observed. The same statistical differences in the pharmacokinetic parameters were observed in the group with 80% hepatectomy. Transplantation of isolated hepatocytes into the spleen did not correct hepatocellular insufficiency. In this study, numerous laboratory tests were performed. A significant correlation was found between serum albumin, cholesterol, factor V, ALAT, total bilirubin, and ratio of amino acids and the pharmacokinetic parameters of antipyrine.

High-performance liquid chromatographic determination of cis-dichlorodiammineplatinum(II) in plasma ultrafiltrate.

A reproducible, simple and sensitive high-performance liquid chromatographic method was described for the quantitative analysis of cis-diamminedichloroplatinum(II) (CDDP) in ultrafiltrate plasma in the presence of nickel chloride as internal standard. CDDP and the internal standard were chelated by exchange with diethyldithiocarbamate. After derivatization, the mixture was directly injected into the column. Chromatography was performed on an Ultrasphere column and the eluent measured spectrophotometrically at 260 nm for CDDP and at 250 nm for the internal standard. The peak area ratio of CDDP to the internal standard varied linearly with concentration over the range 0.05-10 micrograms ml-1. Precision and reproducibility were both excellent and the limit of quantification was 0.03 micrograms ml-1 using only 0.5 ml of ultrafiltrate. The present method, without extraction, should be entirely automated. This assay may be suitable for therapeutic drug monitoring in patients receiving CDDP.

Evaluation of the effects of ambroxol on the ofloxacin concentrations in bronchial tissues in COPD patients with infectious exacerbation.

Infectious excerbations of COPD are generally due to Streptococcus pneumoniae, Haemophilus species, and other Gram-negative bacteria. Ofloxacin has potent activity against Gram-negative species but is less effective against Gram-positive species including Streptococcus pneumoniae. It has also been shown that the administration of ambroxol increases the concentration of some antibiotics in pulmonary tissues. The aim of the study was to determine whether ambroxol increases the bronchial tissue concentrations of ofloxacin to a level exceeding the MIC90 of the bacterial species less susceptible to ofloxacin. 24 patients with COPD were randomized in two groups. Drug regimens of ofloxacin alone (200 mg twice daily) or ofloxacin (200 mg twice daily)+ambroxol (30 mg thrice daily) were administered over 10 d. A fibroscopy was performed on day 10 with bronchial biopsies and broncho-alveolar lavage. At steady state, concentrations of drug in plasma and bronchial samples were assayed by HPLC with fluorometric detection. There was no significant difference in the bronchial levels of ofloxacin between the two groups; however, in alveolar cells, ofloxacin concentration was three times higher in the group with ambroxol. Ambroxol does not increase ofloxacin concentrations in bronchial tissue because high concentrations are already present in the lung.

An enzymatic method for assaying sulbactam in human serum: comparison with high performance liquid chromatography.

An enzymatic method using nitrocefin as substrate was developed to assay sulbactam in human serum. Serum containing sulbactam was incubated with purified titrated TEM-1 beta-lactamase and nitrocefin was then added to the mixture to determine the remaining beta-lactamase activity and consequently the concentration of sulbactam. Assays were carried out on five patients with pulmonary infections receiving sulbactam plus amoxycillin iv. The values for serum sulbactam concentrations determined by the enzymatic method were compared with those determined by high performance liquid chromatography (HPLC). The correlation coefficient was 0.990 for serum sulbactam concentrations below 15 mg/L.

Bayesian estimation of p-aminohippurate clearance by a limited sampling strategy.

This study describes a methodology to calculated p-aminohippurate (PAH) clearance (CL) and volume of distribution (V) with both the population parameters and one or two samples taken during the disposition and the elimination phase after a single intravenous infusion. The computer program P-PHARM was used, and a log-normal distribution and a heteroscedastic residual error distribution were assumed. Ninety-six patients with and without renal insufficiency were available for analysis, and a two-compartment model was used for data modeling. Population parameters were evaluated for 70 patients (mean number of observed concentration per individual, 6) by a three-step approach. In step 1, the computer program was used to estimate the average pharmacokinetic parameters without taking into account the demographic and/or biological factors. In step 2, the relationship between the posterior individual estimates and the covariables was investigated with multiple linear stepwise algorithm. In step 3, the population parameters were re-estimated considering the relationship with the covariables. From the regression performed in step 2, the following covariables were included: serum creatinine, body surface area, and body weight. The population averages of CL and V were 30.7 +/- 2.36 L/h and 10.6 +/- 1.29 L, respectively. To evaluate the predictive performance of the population parameters, the remaining 26 patients were used. The population parameters combined with one or two individual PAH plasma concentrations led to a bayesian estimation of individual CL and V. This estimation was compared with the classical procedure of parameter estimation (individual fitting from multiple blood samples).(ABSTRACT TRUNCATED AT 250 WORDS)

Methotrexate concentrations in synovial membrane and trabecular and cortical bone in rheumatoid arthritis patients.

OBJECTIVE: To determine methotrexate (MTX) concentrations in the synovial membrane (SM) and cortical and trabecular bone of rheumatoid arthritis (RA) patients. METHODS: Ten RA patients (9 women, 1 man; mean +/- SD age 49.2 +/- 10.6, mean disease duration 13.2 +/- 9.9 years) undergoing surgical procedures for rheumatoid articular lesions participated in this study. Mean +/- SD MTX treatment duration was 26.4 +/- 21.3 months. The day preceding surgery, 10 mg of MTX was administered intramuscularly. During surgery, a mean +/- SD of 19.7 +/- 2.6 hours after MTX administration, SM, bone fragments, and blood were collected simultaneously. MTX was assayed by fluorescence polarization immunoassay in plasma and tissues. RESULTS: The mean +/- SD plasma concentration was 0.0252 +/- 0.01 nmoles/ml at the time of tissue sampling. The mean MTX concentration in SM was 0.285 +/- 0.159 nmoles/gm. The mean MTX concentrations in trabecular and cortical bone were 0.292 +/- 0.164 and 0.286 +/- 0.126 nmoles/gm, respectively. CONCLUSION: After intramuscular administration, high MTX concentrations are found in SM and cortical and trabecular bone of RA patients.

Pefloxacin clinical pharmacokinetics.

Pefloxacin has a broad spectrum of activity against a great number of Gram-negative and Gram-positive bacteria. It is also capable of penetration into cells, yielding high tissue:serum ratios, with implications for the treatment of infections caused by intracellular pathogens. Pefloxacin is well absorbed from the gastrointestinal tract. Its elimination half-life ranges from 6.2 to 12.4 hours. After repeated administration, a major change in pharmacokinetic parameters is observed. Pharmacokinetic parameters are minimally altered or not altered in patients with impaired renal function. Altered plasma pharmacokinetics in patients with liver insufficiency and in elderly patients are observed, so dosage adjustments are necessary. In addition, pefloxacin interacts with a number of other compounds at hepatic (e.g. theophylline and cimetidine) and gastrointestinal (e.g. antacids) sites. With the exception of saliva, cerebrospinal fluid, aqueous humor, vitreous fluid and amniotic fluid, body fluid concentrations reach plasma concentrations. Studies on tissue penetration show that concentrations exceeding plasma concentrations are obtained in most tissues. The highest tissue:plasma concentration ratios are achieved in lung and kidney, whereas concentrations in fat are considerably lower than those in plasma.

A reproducible, simple and sensitive HPLC assay for determination of ofloxacin in plasma and lung tissue. Application in pharmacokinetic studies.

A high-performance liquid chromatographic method with fluorometric detection was developed for the analysis of ofloxacin in plasma and lung tissue. The detection was performed at 280 nm for excitation and 500 nm for emission. The procedure involves the addition of an internal standard followed by treatment of the samples with acetonitrile and dichloromethane. The proposed technique is reproducible, selective, reliable and sensitive. Linear detector response was observed for the calibration curve standards in the range of 0.1-5 micrograms ml-1 for plasma and 0.025-2.5 micrograms g-1 for lung tissue. The limit of quantitation is 5 ng ml-1 or 5 ng g-1. The accuracy of the method is good; that is, the relative error is < 10%. This method was applied to the pharmacokinetic study of ofloxacin in 24 chronic obstructive pulmonary disease patients.

Multiple-dose pharmacokinetics of ceftibuten after oral administration to healthy volunteers.

The pharmacokinetics of ceftibuten in plasma and urine were investigated after oral administration. Twelve healthy subjects were treated orally twice daily with 400 mg of the drug for 7 days; on day 8, the subjects received a last dose of 400 mg of ceftibuten. Ceftibuten and its metabolite, the trans isomer of ceftibuten, were assayed in plasma and urine by a specific HPLC method with UV detection. Ceftibuten was rapidly absorbed, as evidenced by the mean time to the maximum observed cis-ceftibuten concentration of 2.4 h. To describe the drug intake process, a Weibull model was used. For the metabolite, the mean time to maximum concentration in plasma was 3.25 h. Mean values for the terminal half-life in plasma were 2.17 h for cis-ceftibuten and 3.19 h for trans-ceftibuten. The overall elimination half-life, tmax, and total and renal clearances of cis-ceftibuten were invariant with respect to duration of dosing. The area under the plasma concentration versus time curve from 0 to infinity and the Cmax of this drug were significantly higher on day 8 than the values predicted from the elimination half-life computed on day 1 of treatment and the dosing interval. The pharmacokinetic parameters of trans-ceftibuten were invariant with respect to duration of dosing. Ceftibuten was well tolerated; there were no clinically significant adverse clinical events. The results from the present study indicate that the levels of cis-ceftibuten in plasma as well as in urine remain above the MICs for susceptible organisms over the dosing interval.