PRL antiapoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity.

During gestation, the uterus undergoes severe changes to accommodate and protect the developing conceptus. In particular, stromal endometrial cells proliferate and differentiate to form the decidual tissue, which produces PRL. Once the conceptus begins to grow, extensive regression by apoptosis take place in the decidua coincident with the loss of the PRL receptor in this tissue. In this report we have established for the first time that PRL, acting through the long form of the PRL receptor and the PI3K pathway, exerts an antiapoptotic effect in rat decidua. We have also shown that protein kinase B phosphorylation on serine 473 as well as its nuclear translocation are stimulated by PRL in decidual cells. Moreover, we have found that caspase-3, a well known effector of apoptosis, becomes expressed and active in the rat decidua just at a time when this tissue undergoes extensive apoptosis. PRL was able to down-regulate both caspase-3 mRNA levels as well as activity. Furthermore, using a protein kinase B dominant-negative expression vector, we provide evidence that PRL inhibition of caspase-3 requires an intact protein kinase B pathway. Finally, we have also found that rat placental lactogen I and II dose-dependently inhibit caspase-3 mRNA, suggesting multiple sources of PRL in the hormonal control of rat decidual regression. In summary, the results of this study have defined an important role for decidual PRL in the normal progress of pregnancy, specifically in the regression and reorganization of the decidua.

Estrogen receptors alpha and beta in rat decidua cells: cell-specific expression and differential regulation by steroid hormones and prolactin.

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), alpha and beta. In this investigation, we analyzed the expression of decidual ERalpha and ERbeta, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERp to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERalpha and ERbeta by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERbeta messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERalpha expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERalpha and had no synergistic effect on ERbeta expression. The use of GG-AD cells, which express only ERbeta, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERbeta is expressed in addition to ERalpha in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERalpha, but concomitantly increases the expression of a functional ERbeta that mediates estradiol up-regulation of the decidual progesterone receptor.

Rat decidual prolactin. Identification, molecular cloning, and characterization.

Establishment and maintenance of pregnancy require the activity of a highly specialized maternal tissue, the decidua. It is well established that the human decidua synthesizes and releases prolactin. However, in the rat, no study has been able to demonstrate the production of prolactin by the decidua. In this report, we established for the first time using Northern blot analysis and reverse transcription-polymerase chain reaction, Western blot analysis, immunocytochemistry, and enzyme-linked immunosorbent assay, that a defined cell population located in the rat antimesometrial decidua expresses prolactin mRNA, as well as synthesizes and secretes this hormone. By reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, we cloned a full-length cDNA for rat decidua prolactin, whose sequence was identical to that of pituitary prolactin. Our results also showed that pituitary prolactin appeared to down-regulate decidual prolactin levels. Under these circumstances, inhibition of pituitary prolactin secretion led to a rise in both decidual prolactin mRNA and protein expression. Moreover, addition of exogenous prolactin to primary decidual cells in culture also caused a marked decrease in decidual prolactin mRNA expression. Finally, treatment of primary decidual cells with steroid hormones or 8-bromo-cAMP revealed a differential regulation of decidual prolactin expression from that of pituitary suggesting a tissue-specific regulation of prolactin gene expression, possibly through the use of an alternative promoter in rat decidua.

The expression of interleukin-6 (IL-6), IL-6 receptor, and gp130-kilodalton glycoprotein in the rat decidua and a decidual cell line: regulation by 17beta-estradiol and prolactin.

The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, progesterone, or PRL. Progesterone showed no effect, whereas estradiol and PRL reduced the level of IL-6 mRNA expression. To examine the mechanism by which these hormones inhibit IL-6 expression, we used a simian virus 40-transformed decidual cell line (GG-AD), which expresses only estrogen receptor-beta (ERbeta). Like primary decidual cells in culture, GG-AD cells express IL-6, IL-6R, and gp130 mRNA. When cultured in the presence of estradiol (0-100 ng/ml), mRNA for IL-6 and its receptor components were down-regulated in a dose-dependent manner. Estradiol also caused a dose-dependent decrease in IL-6 protein secretion into the culture medium. The inhibitory effect of estradiol on IL-6 mRNA expression was reversed by the antiestrogen ICI-164,384. Similar inhibition of IL-6 and gp130 mRNA expression was observed with PRL treatment. However, PRL had no effect on IL-6R mRNA levels. PRL inhibition of IL-6 expression was totally reversed by tyrphostin AG490, a JAK2 inhibitor. In summary, the results of this investigation indicate that IL-6 expression, which is detrimental to the maintenance of pregnancy, is inhibited in the rat decidual tissue. This inhibition is induced by PRL and estradiol, which down-regulate not only IL-6 expression, but also the expression of IL-6 receptor and signaling proteins. The results also suggest that PRL signaling to the IL-6 gene is mediated through the long form of PRL receptor and involves JAK2 activation, whereas that of estradiol can be transduced by estrogen receptor-beta.

Identification of Streptococcus faecalis and Streptococcus faecium and susceptibility studies with newly developed antimicrobial agents.

Identification and susceptibility studies were performed on 301 blood and urine Streptococcus faecalis and Streptococcus faecium isolates. Strep Trio-Tubes S4, S5, and S3 (Carr-Scarborough Microbiologicals, Inc.) were compared with conventional methods for accuracy and rapidity. Of 282 isolates identified as S. faecalis, 98% were identified by species in 4 h with Trio-Tubes; the same percentage of isolates analyzed by conventional methods were identified in 24 h. All 14 S. faecium isolates (approximately 5% of the total number of isolates) were identified by Trio-Tubes in 24 h. In vitro MIC susceptibility testing of the isolates was performed by the Dynatech 2000 microdilution technique (Dynatech Laboratories, Inc.). Several newly developed antimicrobial agents, including imipenem (a carbapenem) and some of the quinolone drugs, i.e., CI-934, ciprofloxacin, A-56619, A-56620, amifloxacin, norfloxacin, and enoxacin, were tested, as were ampicillin, erythromycin, and vancomycin. Both ampicillin and vancomycin showed good activity against S. faecalis, with MICs for 90% of isolates tested (MIC90S) of 1 and 2 micrograms/ml, respectively; with S. faecium, ampicillin exhibited an MIC90 of 16 micrograms/ml and vancomycin exhibited an MIC90 of 2 micrograms/ml. Of the newer antimicrobial agents, imipenem and CI-934 exhibited the greatest activity against S. faecalis strains, with MIC90S of 2 and 0.5 micrograms/ml, respectively. MBCs against the isolates were determined with CI-934, with 90% of S. faecalis strains showing MBCs of 1 microgram/ml or less.

Activated charcoal and resin haemoadsorbents: in vitro drug removal and blood compatibility studies.

An investigation has been made of the drug removal properties and blood compatibility of activated charcoal and resin haemoadsorbents . The adsorbents were Norit RBX1 peat-based extruded charcoal, spherical charcoal as used in the Sorin Biomedica Detoxyl 2 column and 2 cross-linked polystyrene-based macroreticular resins. Drug removal properties were assessed in terms of in vitro adsorption efficiency of each adsorbent for 11 different drugs. The blood compatibility of the charcoals and macroreticular resins was evaluated in an in vitro haemoperfusion circuit by measuring the changes in platelet and leucocyte levels in human blood caused by contact with the adsorbent. The results of the investigation indicate advantages in using spherical charcoal haemoadsorbents .

In vitro assessment of charcoal and resin hemoadsorbents.

An in vitro assessment has been made of the blood compatibility and efficiency of different charcoal and resin hemoadsorbents. The charcoals vary in source, shape, size, nature and amount of polymer coating. The resins are the macroreticular adsorbents XAD-4 and Y56 and a carbonized resin. The effect on Y56 of albumin coating has been studied. The assessment has demonstrated that a reduction in platelet drop is accompanied by a decrease in the adsorption of fibrinogen. The results confirm the potential advantages of spherical charcoal and that coating with an ultrathin layer of polymer or with albumin does not seriously reduce efficiency. For Y56 resin, treatment with an albumin solution of 1-2 g/100 ml appears to be a suitable procedure if clinical use is considered.