Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis.

Endometriosis is a disease characterized by the localization of endometrial tissue outside the uterine cavity. The differences observed in migration of human endometrial stromal cells (hESC) obtained from patients with endometriosis versus healthy controls were proposed to correlate with the abnormal activation of Raf-1/ROCKII signalling pathway. To evaluate the mechanism by which Raf-1 regulates cytoskeleton reorganization and motility, we used primary eutopic (Eu-, n = 16) and ectopic (Ec-, n = 8; isolated from ovarian cysts) hESC of patients with endometriosis and endometriosis-free controls (Co-hESC, n = 14). Raf-1 siRNA knockdown in Co- and Eu-hESC resulted in contraction and decreased migration versus siRNA controls. This phenotype was reversed following the re-expression of Raf-1 in these cells. Lowest Raf-1 levels in Ec-hESC were associated with hyperactivated ROCKII and ezrin/radixin/moesin (E/R/M), impaired migration and a contracted phenotype similar to Raf-1 knockdown in Co- and Eu-hESC. We further show that the mechanism by which Raf-1 mediates migration in hESC includes direct myosin light chain phosphatase (MYPT1) phosphorylation and regulation of the levels of E/R/M, paxillin, MYPT1 and myosin light chain (MLC) phosphorylation indirectly via the hyperactivation of ROCKII kinase. Furthermore, we suggest that in contrast to Co-and Eu-hESC, where the cellular Raf-1 levels regulate the rate of migration, the low cellular Raf-1 content in Ec-hESC, might ensure their restricted migration by preserving the contracted cellular phenotype. In conclusion, our findings suggest that cellular levels of Raf-1 adjust the threshold of hESC migration in endometriosis.

A transitive perspective on the relief of psychosomatic symptoms.

A key element of successful psychotherapy for the treatment of psychosomatic disorders is that patients recognize and change the meaning of their experiences. Such changes are brought about by appropriate verbal referencing of symptoms currently experienced within a given narrative. The present theoretical paper argues that changes are not based on better, more adaptive narratives per se, but on the transition (or linkage) process itself that is experienced between different narratives. This view is theoretically justified in various ways: first, it is accounted for through contemporary spatiotemporal neuroscience, which aims to connect mental and structural aspects via a common dynamic property or, according to Northoff, the "common currency" of a brain's orientation along its embeddedness in its contextual world, i.e., body and environment. Second, it is justified through the physics concept of "spontaneous symmetry breaking," which is used analogously to "suffering from symptoms." If the sufferer is willing to experience a process of "going back," that is, moving away from the previous narrative (or aspect) by verbally relating to the felt aspects of the symptom in question (i.e., approaching its meaning), they are moving toward symmetry or an underlying dynamic alignment with their world context. Clinical predictions are derived from the theoretical arguments.

Testosterone induced downregulation of migration and proliferation in human Umbilical Vein Endothelial Cells by Androgen Receptor dependent and independent mechanisms.

Recent research has emphasized the potential unfavorable effects of declining testosterone (T) levels in men and the putative beneficial effect of androgen therapy in select women. Some controversy surrounding the mechanism of action and the effects of T on endothelium remains. In this study, we evaluated the mechanism of T action on pooled primary Human Umbilical Vein Endothelial Cells (HUVEC) of mixed gender by focusing on two important processes, proliferation and migration. In our in vitro model system, we found that only the supra-physiological dose of T affected these two processes irrespective of the ratio of male to female cells in the pools. At a concentration of 1 µM, T downregulated the proliferation of HUVEC by inducing arrest in the G1 cell cycle phase in an Androgen Receptor (AR)-independent manner. We show that treatment with 1 µM T also induced downregulation of HUVEC migration. This process was AR-dependent and was associated with persistent phosphorylation of ezrin, radixin and moesin. Regardless of the mechanism of action, the treatment of HUVEC with both supra- and physiological doses of T was associated with posttranscriptional stabilization of the AR upon ligand binding.

The estrogen metabolite 17beta-dihydroequilenin counteracts interleukin-1alpha induced expression of inflammatory mediators in human endothelial cells in vitro via NF-kappaB pathway.

In most studies showing cardio- and vasculoprotective effects of estrogens, 17beta-estradiol was used and little information on possible effects of different estrogen metabolites is yet available. We investigated whether particular estrogen metabolites are effective in counteracting inflammatory activation of human endothelium. Human endothelial cells were incubated with 17alpha-dihydroequilenin, 17beta-dihydroequilenin, delta-8,9-dehydroestrone, estrone and 17beta-estradiol and stimulated with interleukin (IL)-1alpha. The expression of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) was determined. 17beta-dihydroequilenin and 17beta-estradiol at a concentration of 1 microM reduced IL-1alpha-induced up regulation of IL-6, IL-8 and MCP-1 close to control levels. When both compounds were used in combination an additive effect was observed. 17alpha-dihydroequilenin and delta-8,9-dehydroestrone showed a similar anti-inflammatory effect only when used at 10 microM whereas estrone had no effect. The effect of 17beta-dihydroequilenin on IL-1alpha-induced production of IL-6, IL-8 and MCP-1 was reversed by the estrogen receptor antagonist ICI 182,780. 17beta-dihydroequilenin also inhibited IL-1alpha-induced translocation of p50 and p65 to the nucleus of the cells. We have identified the estrogen metabolite 17beta-dihydroequilenin, as an inhibitor of inflammatory activation of human endothelial cells. Characterization of specific estrogens--as shown in our study--could provide the basis for tailored therapies, which might be able to achieve vasoprotection without adverse side effects.

Induction of labor at term using isosorbide mononitrate simultaneously with dinoprostone compared to dinoprostone treatment alone: a randomized, controlled trial.

OBJECTIVE: We aimed to determine whether isosorbide mononitrate (IMN) given simultaneously with dinoprostone in term pregnancies is superior to dinoprostone alone to promote delivery. STUDY DESIGN: One hundred and twenty nulliparous women at term were randomly assigned to receive per vaginam IMN 40 mg or placebo in addition to 3 mg dinoprostone 2 times daily for up to 2 days. Analysis was by intention to treat. RESULTS: Baseline characteristics of both groups were comparable. The induction to delivery intervals did not differ between the IMN and the placebo group (26.4 +/- 14.4 vs 23.4 +/- 14.8 hours, P = .408). IMN resulted in more headache compared to placebo (32/55 [58.2%] vs 2/55 [3.6%], P < .001). CONCLUSION: Vaginally administered IMN does not play a role in promoting delivery in term pregnancy if given at the same time as dinoprostone. This might reflect its relaxant effect on the uterine fundus, which may overcome its cervical softening effect.

Differential regulation of proteasome-dependent estrogen receptor alpha and beta turnover in cultured human uterine artery endothelial cells.

Estrogen-induced loss of estrogen receptor (ER) alpha expression limits estrogen responsiveness in many target cells. However, whether such a mechanism contributes to changes in vascular endothelial ER alpha and/or ER beta levels is unclear. Using RT-PCR assays, we did not find any regulation of ER alpha or ER beta mRNA expression in human uterine artery endothelial cell (HUAEC) nuclear extracts on stimulation with 17 beta-estradiol for 1 or 2 h. By contrast, Western analysis on HUAEC extracts revealed that 17 beta-estradiol was capable of down-regulating both ER alpha and ER beta protein starting 1 h after treatment, an effect that can be blocked by pretreatment with tamoxifen as well as with the proteasome inhibitor lactacystin. The proteolysis inhibitors insulin, cycloheximide, and puromycin impede ER alpha, but not ER beta, turnover. Ubiquitin, but not its competitive inhibitor methyl-ubiquitin, induces rapid turnover of both ERs in a cell-free system of MCF-7 and HUAEC extracts. We, thus, propose the existence of estrogen-induced ER degradation that serves to control physiological responses in an estrogen target tissue, i.e. human vascular endothelium, by down- regulating ER alpha as well as ER beta through different proteasomal uptake mechanisms.

Identification of estrogen and progesterone receptor mRNA expression in the conjunctiva of premenopausal women.

PURPOSE: The purpose of the present study was to identify the expression of estrogen and progesterone receptor mRNA and of estrogen and progesterone receptor protein in the conjunctiva of healthy women. METHODS: Specimens of conjunctival tissue of 10 premenopausal women (age range, 13-38 years) were obtained during ophthalmic surgery in patients under general anesthesia. Specimens of approximately 4 mm(2) were taken from superior, nasal, or temporal bulbar conjunctiva adjacent to the bulbus and were immediately deep frozen with liquid nitrogen. Four women underwent strabismus surgery, two had phacoemulsification, and four had vitrectomy. Only three women were taking oral contraceptives. The expression of estrogen receptor (ER)-alpha, ERbeta, and progesterone receptor (PR) mRNA was analyzed by RT-PCR. Western blot analysis on nuclear extracts was performed with the anti-ERalpha mouse monoclonal antibody AB-15, the anti-ERbeta mouse monoclonal antibody 6B12, and the anti-PR mouse monoclonal antibody PgR 636. RESULTS: In two samples, ERalpha, ERbeta, and PR mRNAs were not accessible because of highly degraded RNA. In the remaining eight samples, an appearance rate of 100% was obtained for all three mRNAs. Similarly, an appearance rate of 100% was obtained for ERalpha, ERbeta, and PR protein in nine tissue samples accessible for analysis; one sample could not be analyzed due to a low amount of tissue. CONCLUSIONS: This study confirms the existence of estrogen and progesterone receptors in the human conjunctiva of premenopausal females. Because the proteins of estrogen and progesterone were also found in this study's specimens, the data indicate that the conjunctiva is a target site for sex steroids. Future studies are needed to elucidate the role of these receptors in ocular diseases involving the conjunctiva.

Treatment of functional hypothalamic amenorrhea with hypnotherapy.

OBJECTIVE: To determine the effects of hypnotherapy on resumption of menstruation in patients with functional hypothalamic amenorrhea (FHA). DESIGN: Uncontrolled clinical study. SETTING: Academic clinical care center. PATIENT(S): Twelve consecutive women with FHA were selected. INTERVENTION(S): A single 45- to 70-minute session of hypnotherapy was administered, and patients were observed for 12 weeks. MAIN OUTCOME MEASURE(S): Patients were asked whether or not menstruation resumed and whether or not well-being and self-confidence changed. RESULT(S): Within 12 weeks, 9 out of 12 patients (75%) resumed menstruation. All of the patients, including those who did not menstruate, reported several beneficial side effects such as increased general well-being and increased self-confidence. CONCLUSION(S): Hypnotherapy could be an efficacious and time-saving treatment option that also avoids the pitfalls of pharmacological modalities for women with FHA.

Quantitative determination of carbon monoxide in cell culture supernatants by spectrophotometric analysis.

A simple, sensitive, nonradioactive method for measuring carbon monoxide (CO) in cell culture supernatant is described. Dissolved CO reacts with hemoglobin (Hb) to carboxyhemoglobin (HbCO) in a modified Conway cell. HbCO is quantified by spectrophotometric analysis, and total concentration of CO given in microg CO/l cell culture supernatant is mathematically calculated. Furthermore, we compared our newly developed method with a recently published method. Confluent human umbilical vein epithelial cells (HUVEC) were incubated with 10(-6) M hydrocortisone known to induce heme oxygenase-2 protein and transcript expression for 4 h and CO production was measured. Levels following hydrocortisone treatment were significantly enhanced compared to controls when using our newly developed technique (p<0.05), whereas only a nonsignificant trend could be observed using the recently published method. We conclude that this nonradioactive technique to quantify CO is more sensitive than previous ones, thereby allowing to measure even physiologic quantities of CO in aqueous solutions.

Testosterone dependent androgen receptor stabilization and activation of cell proliferation in primary human myometrial microvascular endothelial cells.

OBJECTIVE: To clarify, whether uterine endothelial proliferation could be regulated via an autocrine estrogen producing mechanism or direct actions of testosterone. DESIGN: In vitro study. SETTING: Tertiary care facility. PATIENT(S): Human myometrial tissue obtained from 40 women undergoing hysterectomy without further intrauterine pathology. INTERVENTION(S): Cell culture, proliferation assay and CYP19 activity assay on human myometrial endothelial cells treated with testosterone, estradiol, letrozole, flutamide, PD98059, MG-132 alone or in combination. MAIN OUTCOME MEASURE(S): We analyzed whether aromatase is expressed in human myometrial microvascular endothelial cells (HMMECs) and whether it affects proliferation and converts androgens to estrogens. In addition, we aimed to define whether or not T could have a direct capability to affect HMMEC proliferation. RESULT(S): Using quantitative real-time PCR and Western analysis, primary passage four HMMECs were shown to express low levels of aromatase mRNA and protein, respectively. However, HMMECs were unable to convert radioactively labeled 3∗H-1ß-androstenedione to estrogen. Pharmacologic doses of T (10(-6) and 10(-4) M) increased HMMEC proliferation, assessed through a bromodeoxyuridine ELISA. This effect of T on proliferation could not be blocked after pretreatment of cells with the aromatase inhibitor letrozole. In addition, HMMECs were found to express androgen receptors (ARs), and the AR antagonist flutamide abolished T-dependent proliferation. T was shown to increase AR protein levels, which was due to T-dependent receptor stabilization and not activation of gene transcription. CONCLUSION(S): We conclude that myometrial endothelial proliferation is not regulated through myometrial endothelial estrogen production. However, pharmacologic doses of T increase myometrial endothelial proliferation through a receptor-dependent and -stabilizing mechanism.

Paradoxical hypnotic experiences in escaping constraining dilemmas: a clinical example.

Deciding how to choose from opposing options often seriously impacts people's final selections. Such constraining options are frequently associated with feelings of hopelessness, depression, or chronic pain. As an example of such situations, a model is presented with material from a single case that utilized previous contradictory experiences in the treatment of a woman patient who suffers from chronic pelvic pain. The case summarizes how previous experiences, which have been paradoxical, can serve as substrates of behavioral change, which in turn can emerge in a way that allows the patient to integrate these experiences, personally and slowly, without conscious effort.

Estrogen receptor-beta is the predominant estrogen receptor subtype in normal human synovia.

OBJECTIVE: Joint pain increases after menopause with more than 50% of woman suffering from arthralgies. Since pain and inflammation of joints originate from synovial tissue, we aimed to discover whether estrogen receptors are present in the human synovia. METHODS: This in vitro study was performed on samples of human synovial tissue, obtained from pre- (n = 8) and postmenopausal woman (n = 11) and men (n = 5) following surgery due to traumatic lesions. Fresh synovial tissue specimens were assessed for the localization as well as the presence of estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta) by means of immunohistochemistry, as well as Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: ER beta protein and mRNA were found to be equally and highly expressed in synovial stroma and lining cells of all explants independent of sex or menopausal status. In contrast, weak ER alpha staining was localized in the synovial lining cells in only three of 24 explants. ER alpha protein was found to be weakly expressed in three of ten explants. ER alpha mRNA was found with highly variable amounts in seven of ten explants. CONCLUSION: In view of our observation that ER beta but not ER alpha is expressed regularly in normal human synovia in high amounts, we propose that estrogen could play a significant role in synovial membrane function in women and men, operating preferably via the ER beta isoform.

Second trimester abortion using isosorbide mononitrate in addition to gemeprost compared with gemeprost alone: a double-blind randomized, placebo-controlled multicenter trial.

OBJECTIVE: We aimed to determine whether second-trimester abortion using isosorbide mononitrate (IMN) in addition to gemeprost is more effective and reduces side effects compared with gemeprost alone. STUDY DESIGN: Eighty women who were age 13 to 23 weeks' gestation were randomly assigned to receive per vaginam either IMN 40 mg (group 1, 40 women) or placebo (group 2, 40 women) in addition to gemeprost 1 mg up to 3 times daily 3 hours apart for 2 days. Analysis of variance, a chi 2 test, and a multivariate analysis were performed. RESULTS: Of the 72 women analyzed, 68% (group 1) and 38% (group 2) underwent abortion within day 1 (P < .05). However, group 1 was associated with more headache (18% of women) 3 hours after induction compared to group 2 (0% of women, P = .038). CONCLUSION: IMN in addition to gemeprost is effective for second-trimester abortion, but is associated with more headache compared with gemeprost alone.