Role of prolactin and glucocorticoids in the expression of casein genes in rabbit mammary gland organ culture. Quantification of casein mRNA.

Milk synthesis is initiated solely by prolactin in the pseudopregnant rabbit and glucocorticoids potentiate this action of prolactin. In organ culture, prolactin, in the presence or in the absence of insulin, enhances casein synthesis and cortisol (inactive alone) amplifies this action. Measurements of casein mRNA concentration in total cellular RNA, by hybridization with DNA complementary to casein mRNA, revealed that the stimulation of casein synthesis by the glucocorticoid is accompanied by an increase in the amount of casein mRNA. A systematic comparison of variations of these two parameters indicated that the major effect of glucocorticoids on lactogenesis in the rabbit at this stage of mammary gland development is mediated through an increase in the quantity of casein mRNA available for translation. No simultaneous control of casein mRNA translation by cortisol was observed.

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells.

OBJECTIVE: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS: Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS: Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.

Evolution of prolactin and placental lactogen receptors in ewes during pregnancy and lactation.

The present paper describes a method of membrane preparation from ewe mammary gland using a sucrose cushion (1.3 M) to select smooth membranes; this results in a membrane preparation richer in PRL receptors than the microsomal preparation classically used. This method was used for the characterization and measurement of PRL and ovine placental lactogen (oPL) receptors in three organs of the ewe (mammary gland, liver, and adipose tissue). PRL receptors were measured by competition of iodinated human GH ([125I]hGH) with ovine PRL (oPRL). This hormone, which has both growth and lactogenic activities, appears to interact with PRL receptors with a higher affinity than oPRL itself and is a good probe for the determination of PRL receptors in the ewe. oPL receptors were measured by the specific binding of [125I]oPL. This hormone appears to bind exclusively to a somatogenic site in the ewe, since various GHs compete efficiently for binding, whereas oPRL is without effect. The evolution of PRL and oPL receptors was determined during pregnancy and lactation and at different periods after an estradiol and progesterone treatment, which provokes growth of the mammary gland and milk secretion. During pregnancy, PRL receptors increased in the mammary gland up to day 100. During the last trimester, receptor content remained stable, and a second increase occurred during early lactation. No additional significant changes were observed either for PRL receptors in liver or adipose tissue or for oPL receptors in any of the organs studied (mammary gland, liver, adipose tissue). Injections of large doses of estradiol and progesterone to nonpregnant ewes were able to reproduce effectively the pattern of PRL receptors observed during pregnancy, but had no effect on oPL receptor levels. These studies demonstrate the independence of PRL and PL receptor sites in the ewe and suggest a different hormonal regulation for each type of receptor.

Stabilization of casein mRNA by prolactin and glucocorticoids.

Prolactin injected into pseudopregnant rabbits led to a parallel enhancement of casein synthesis and casein mRNA concentration. When this stimulation was followed by a withdrawal of prolactin obtained by injections of bromocriptine, the rate of casein synthesis progressively diminished. In the presence of endogenous prolactin after the initial stimulation, the decline of casein synthesis was delayed. Hydrocortisone acetate injected with bromocriptine after the initial stimulation by prolactin was able to maintain a high rate of casein synthesis. Measurements of casein mRNA concentration by hybridization with casein cDNA indicated that in all cases the amount of casein mRNA was correlated with the magnitude of casein synthesis. This suggests that the lactogenic hormones, prolactin and glucocorticoids, which were previously demonstrated to be responsible for the enhancement of casein mRNA concentration are involved in their stabilization.

Role of spermidine in casein gene expression in the rabbit.

Spermidine concentration in rabbit mammary gland was estimated during pregnancy, lactation and after the induction of milk synthesis by prolactin and glucocorticoids in vivo and in vitro. It was observed that mammogenesis and lactogenesis during preganancy and the initiation of milk secretion at parturition are accompanied by an enhancement of spermidine concentration in the mammary gland. By contrast, the initiation of these phenomena by hormone injections does not require such variations of spermidine concentration. In organ culture, a slight increase in spermidine concentration was obtained under the influence of an hormonal combination including insulin, prolactin and cortisol. Spermidine added to the culture medium was unable to mimic cortisol action. An amplification of casein synthesis and a parallel increase of casein mRNA concentration was provoked by cortisol even when spermidine synthesis was blocked. Thus, one of the major actions of glucocorticoids during lactogenesis in the rabbit is not mediated through an increase in spermidine concentration in the mammary gland.

Stimulation of milk synthesis in the rabbit by fish pituitary extract.

The lactogenic properties of extracts of the pituitary glands of salmon and trout were evaluated by using the organ culture technique with rabbit mammary explants. Crude extracts and fractions obtained after chromatography on Ultrogel and selected for their capacity to compete with ovine prolactin in a rabbit mammary gland radioreceptor assay were added to the culture medium. The criteria of lactogenesis were lactose synthetase activity, casein synthesis, measurements of the concentration of casein messenger RNA and the histology of mammary glands. All these tests led to the conclusion that salmon and trout pituitary glands contain a prolactin-like principle capable of initiating milk synthesis in the rabbit mammary cell.

Prolactin receptor turnover in explants of pseudopregnant rabbit mammary gland.

Pseudopregnant rabbit mammary glands in organ culture were used to investigate prolactin (PRL) receptor turnover. Chloroquine (100 microM) results in an increase in prolactin receptor levels (15.7 +/- 1.2% to 35.9 +/- 3.5% specific binding), whereas cycloheximide (1 microgram/ml) induces a rapid decline (to 6.4 +/- 1.2%) suggesting a rapid synthesis and degradation of the receptor molecule. Inhibitors of cellular transcription have little effect on receptor levels. Neither actinomycin D nor dichlororibofuranosylbenzimidazole (DRB) diminish PRL receptor levels whereas total protein synthesis is almost completely inhibited, and chloroquine increases the binding even in the presence of transcriptional inhibitors. These results imply that receptor synthesis continues and that the mRNA for the receptor protein is particularly stable. Ouabain (3 micrometers), which blocks the ATP-dependent Na+/K+ pump, provokes a greater than 60% reduction in PRL receptor levels without modifying total protein synthesis. Dinitrophenol (DNP, 1 mM), an oxidative uncoupler, has little effect on receptor levels, possibly due to a blockage of both synthesis and degradation. Prolactin is capable of inducing a 60% down-regulation of its own receptor, and this phenomenon appears to be energy-dependent because it is partially inhibited by DNP. This process seems to involve an increased rate of receptor degradation. These studies suggest that, at any one time, the level of PRL receptors in a target cell is the result of a dynamic equilibrium between receptor synthesis and degradation and that the most frequent modulations occur at the level of translation and lysosomal degradation. In conclusion, in mammary glands of the pseudopregnant rabbit, the prolactin receptor molecule appears to have a short half-life; the mRNA for this protein, however, is relatively stable.

Fetal levels of tobramycin following maternal administration.

A single dose of tobramycin 2 mg/kg was given intravenously to a woman who presented a 22-week heterotopic pregnancy with both intrauterine and ovarian gestations and two living fetuses. After excision of the adnexum, tobramycin levels were measured in the maternal serum and in the fetal fluids and tissues. Antibiotic levels were especially high in the fetal spleen and kidney.

Transplacental passage of vancomycin during the second trimester of pregnancy.

Pharmacokinetics and drug monitoring of vancomycin were studied at mid-pregnancy in a patient with chorioamnionitis due to Streptococcus agalactiae. The terminal half-life remained in the normal range (4-6 hours) because of an equivalent increase in both volume of distribution and total plasma clearance. Transplacental passage of the drug was observed. Monitoring is mandatory for prolonged vancomycin therapy, and the results should be available within 24 hours. The therapeutic regimen of 15-20 mg/kg every 12 hours was sufficient for this patient's chorioamnionitis. Serum drug levels and renal function should be measured before increasing the vancomycin dosage.

Establishment and characterization of immortalized ovine Sertoli cell lines.

The objective of this study was to generate immortalized Sertoli cell lines from prepubertal lamb testes to facilitate investigations during the course of testicular differentiation. The Sertoli cells were enzymatically isolated and immortalized by transfection, with the sequences coding for the SV40 large T-antigen fused downstream of regulatory elements from the human vimentin gene. The different cell lines were positively stained with antibodies to vimentin and transferrin, in agreement with their Sertoli origin. Reverse transcriptase polymerase chain reaction was used to analyze the specific expression of molecular markers (clusterin/sulfated glycoprotein ISGP-2], follicle-stimulating hormone [rFSH], alpha-inhibin, anti-Müllerian hormone, Wilms' tumor gene [WT-1], steroidogenic factor 1 [SF-1], SRY-related HMG box gene g [SOX9], and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, alpha-inhibin, WT-1, SOX9, and SF-1 (except SF-1 for clone no. 1). Moreover, we performed alkaline phosphatase and receptor tyrosine kinase p145 (c-kit) detection to ensure the absence of contamination by peritubular, germ cells, and Leydig cells. Both tests were negative for all the seven cell lines. These ovine Sertoli cell lines are the first ones obtained from livestock that exhibit specific Sertoli cell characteristics resembling different stages of phenotypic development. They provide useful in vitro model systems for toxicological investigations, coculture, and transfection experiments, making it possible to study signal transduction pathways, cell-cell interactions, and gene expression in species other than rodents.

Pharmacokinetics of cyclosporin A during pregnancy; monitoring of treatment and specific assays of cyclosporin, based on five liver transplant patients.

Very little information is available concerning the pharmacokinetic behaviour and monitoring of cyclosporin A (CsA) during pregnancy, notably after liver transplant. Monitoring of blood levels of CsA is considered to be one of the best tools for evaluation of the efficacy of immunosuppressant treatment. The aim of this study was to bring together new information concerning pregnant women receiving immunosuppressant treatment with CsA and, in view of the special pathophysiological status of such patients, to compare pharmacokinetic profiles of changes in blood levels of CsA and of the combination of CsA plus metabolites. Specific (CsA-S kit) and a new non-specific (CsA-NS kit) assays of CsA were carried out in five hospitalized pregnant patients who had received liver transplants between the 6th and the 41st weeks of amenorrhea. The results of five cases investigated lead to the following conclusions: (1) The pharmacokinetic behaviour of native CsA in the pregnant woman between the 6th and 41st weeks of amenorrhea suggests no systemic accumulation nor any radical need for changes in dosage schedule as compared with a non-pregnant patient. (2) Monitoring based upon simultaneous use of the CsA-NS and CsA-S kits may be a source of analytical bias and hence confusion for the physician. (3) Determination of an experimental CsA-NS/CsA-S accumulation ratio (based upon analysis of single concentrations or processing of AUCs) is of interest only if specific assays involve not only CsA itself but also its principal metabolites. (4) Monitoring based upon single measurements of residual CsA levels only, is necessary and adequate. Furthermore, such an approach is less costly.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of various hormone treatments on induction of lactation in the ewe.

In Exp. I and II, 52 of 68 ewes were induced into lactation with twice-daily injections of estradiol-17 beta (E2-beta) and progesterone (P4; .5 and 1.25 mg/kg body weight/day) for 7 days. Additional treatments were twice-daily injections (days 18 to 20) of hydrocortisone, growth hormone, thyroxine and thyrotropin releasing hormone alone or in various combinations. In Exp. III, 12 ewes were induced into lactation. In this experiment, all ewes were injected with E2-beta and hydrocortisone, as previously, but four ewes (III-2) had P4 injections extended to day 20, and four ewes (III-3) were not injected with P4. Across experiments, lowest milk yields during lactation and the lowest percentage of ewes induced into lactation (58%) occurred when only E2-beta and P4 were injected. Inclusion of hydrocortisone injections (50 mg/day) induced the highest percentage of ewes into lactation (86%, 38 of 44), the highest peak daily yields of milk and the highest total yields during lactation. Including injections of growth hormone, thyroxine or thyrotropin releasing hormone alone or in combinations did not produce better results than injections of E2-beta and P4 alone. Injections of E2-beta and hydrocortisone without concurrent injections of P4 were less effective. Intramuscular injections of P4 (10 mg/day) from days 8 to 20 did not inhibit lactogenesis or subsequent lactation. Across all experiments, 76% of multiparous (52/68) and 50% of nulliparous (6/12) ewes produced greater than 100 ml milk/day during their lactation (34 to 95 days). However, yields of milk for ewes that lactated were only 25 to 50% of those from postpartum ewes. The importance of including injections of hydrocortisone in the induction procedure was established, but determination of optimum time to inject and potential importance of other hormones requires additional research.

[Pharmacokinetics of methotrexate and clinical response associated in the medical treatment of ectopic pregnancies]. / Pharmacocinétique du méthotrexate et réponse clinique associée dans le traitement médical des grossesses extra-utérines.

Methotrexate (MTX) is used in the medical treatment of unruptured ectopic pregnancy. This drug is administered by intramuscular (IM) or intrasaccular (IS) injection under sonographic control. No data are available concerning the pharmacokinetics of MTX in this new indication. This study compares the two administration routes in 12 patients (21 to 37 years) taken as their own control and presenting with ectopic pregnancy (51 +/- 12 days of amenorrhea). Our aim was to compare the pharmacokinetic profile of MTX for each route to facilitate its use in the future. The initial level of hCG was 4.474 +/- 4.184 mIU/ml. Each patient firstly received 1 mg/kg of MTX intrasaccularly under vaginal sonography. The same dose was injected intramuscularly 48 hours later. The pharmacokinetic profiles of MTX after IS and IM administrations were determined after both injections during 48 hours. MTX serum levels were measured by Fluorescence Polarization Immuno Assay. Data were analyzed by model independent methods and compared by a Wilcoxon T test (p 0.01 was considered as significant). All the unruptured ectopic pregnancy were cured and the hCG serum levels were normalized (10 mIU/ml) in 37 +/- 18 days. After IM administration, AUC0-infinity is significantly (p 0.01) increased i.e., 15.1 +/- 4.1 mumol.h/l versus 11.2 +/- 4.8 after IS injection. T1/2 lambda z and MRT remained unchanged whatever the route is i.e., 11.3 +/- 4.9 h and 8.6 +/- 3.9 h (IS) versus 12.1 +/- 5.9 h and 7.3 +/- 1.8 h (IM). The decrement of AUC0-infinity 8 determined after IS injection might be the consequence of the capture of the MTX by the trophoblastic cells (target cells).(ABSTRACT TRUNCATED AT 250 WORDS)

[The administration of tobramycin in the 2nd and 3rd trimester of pregnancy: contribution to a pharmacokinetic study for the adaptation of posology]. / Administration de la tobramycine au cours du deuxième et du troisième trimestre de la grossesse: apport d'une étude pharmacocinétique pour l'adaptation de la posologie.

Aminoglycosides are currently used during pregnancy for the treatment of Staphylococcus, Enterobacteriaceae, Listeria monocytogenes, and Pseudomonas aeruginosa infections. The pharmacokinetics of tobramycin, an aminoglycoside antibiotic, was investigated after a 2.5 mg/kg short intravenous infusion and a once-daily dose regime in 18 pregnant women divided into 2 groups of 9 during the second (Group I: from 20 to 28 weeks of amenorrhoea) and the third (Group II: greater than or equal to 28 weeks of amenorrhoea) trimesters of pregnancy (during these period, risks of infectious diseases are increased). Plasma concentrations of tobramycin were measured by fluorescence polarization immunoassay (FPIA). The decrease of clearance (decrement of 27.6%), at 28 weeks and more gestation leads to an increase in the half-life and the MRT observed in the second group (increment of 49% and 41% respectively), whereas the volume of distribution remained unchanged in the two groups. No accumulation of the drug was observed in pregnant women. Pharmacokinetic disorders are correlated with the duration and moreover with the weight deviation of the women i.e., the growth of the conceptus. In 10 cases, a feto-maternal concentration ratio was calculated at delivery using an umbilical cord blood sample. This findings suggest a phenomenon of accumulation in the conceptus. To limit the potential nephrotoxicity and ototoxicity of tobramycin for the mother and the fetus, a once-daily dose regime seems to be an advanced solution for treatment of nonneutropenic pregnant women.