Estradiol-17␤ (E 2 ) acts through the estrogen receptor (ER) to regulate uterine growth and functional differentiation. To determine whether E 2 elicits epithelial mitogenesis through epithelial ER versus indirectly via ERpositive stromal cells, uteri from adult ER-deficient ER knockout (ko) mice and neonatal ER-positive wild-type (wt) BALB͞c mice were used to produce the following tissue recombinants containing ER in epithelium (E) and͞or stroma (S), or lacking ER altogether: wt-S ؉ wt-E, wt-S ؉ ko-E, ko-S ؉ ko-E, and ko-S ؉ wt-E. Tissue recombinants were grown for 4 weeks as subrenal capsule grafts in intact female nude mice, then the hosts were treated with either E 2 or oil a week after ovariectomy. Epithelial labeling index and ER expression were determined by [ 3 H]thymidine autoradiography and immunohistochemistry, respectively. In tissue recombinants containing wt-S (wt-S ؉ wt-E, wt-S ؉ ko-E), E 2 induced a similar large increase in epithelial labeling index compared with oil-treated controls in both types of tissue recombinants despite the absence of epithelial ER in wt-S ؉ ko-E tissue recombinants. This proliferative effect was blocked by an ER antagonist, indicating it was mediated through ER. In contrast, in tissue recombinants prepared with ko-S (ko-S ؉ ko-E and ko-S ؉ wt-E), epithelial labeling index was low and not stimulated by E 2 despite epithelial ER expression in ko-S ؉ wt-E grafts. In conclusion, these data demonstrate that epithelial ER is neither necessary nor sufficient for E 2 -induced uterine epithelial proliferation. Instead, E 2 induction of epithelial proliferation appears to be a paracrine event mediated by ER-positive stroma. These data in the uterus and similar studies in the prostate suggest that epithelial mitogenesis in both estrogen and androgen target organs are stromally mediated events.

Two chemicals previously shown to have estrogenic activity, bisphenol A and octylphenol, were examined for their effects on accessory reproductive organs and daily sperm production in male offspring of mice fed these chemicals during pregnancy. These chemicals are used in the manufacture of plastics and other products, and have been detected in food and water consumed by animals and people. From gestation day 11-17 female mice were fed an average concentration (dissolved in oil) of bisphenol A or octylphenol of 2 ng/g body weight (2 ppb) and 20 ng/g (20 ppb). The 2 ppb dose of bisphenol A is lower than the amount reported to be swallowed during the first hour after application of a plastic dental sealant (up to 931 micrograms; 13.3 ppb in a 70 kg adult). We found that the 2 ng/g dose of bisphenol A permanently increased the size of the preputial glands, but reduced the size of the epididymides; these organs develop from different embryonic tissues. At 20 ng/g, bisphenol A significantly decreased efficiency of sperm production (daily sperm production per g testis) by 20% relative to control males. The only significant effect of octylphenol was a reduction in daily sperm production and efficiency of sperm production at the 2 ng/g dose. A new approach to studying physiologically relevant doses of environmental endocrine disruptors is discussed, particularly with regard to the development of the reproductive organs, the brain, and behavior.

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Background High blood pressure is common in acute stroke and is a predictor of poor outcome; however, large trials of lowering blood pressure have given variable results, and the management of high blood pressure in ultra-acute stroke remains unclear. We investigated whether transdermal glyceryl trinitrate (GTN; also known as nitroglycerin), a nitric oxide donor, might improve outcome when administered very early after stroke onset. Methods We did a multicentre, paramedic-delivered, ambulance-based, prospective, randomised, sham-controlled, blinded-endpoint, phase 3 trial in adults with presumed stroke within 4 h of onset, face-arm-speech-time score of 2 or 3, and systolic blood pressure 120 mm Hg or higher. Participants were randomly assigned (1:1) to receive transdermal GTN (5 mg once daily for 4 days; the GTN group) or a similar sham dressing (the sham group) in UKbased ambulances by paramedics, with treatment continued in hospital. Paramedics were unmasked to treatment, whereas participants were masked. The primary outcome was the 7-level modified Rankin Scale (mRS; a measure of functional outcome) at 90 days, assessed by central telephone follow-up with masking to treatment. Analysis was hierarchical, first in participants with a confirmed stroke or transient ischaemic attack (cohort 1), and then in all participants who were randomly assigned (intention to treat, cohort 2) according to the statistical analysis plan. This trial is registered with ISRCTN, number ISRCTN26986053.

SummaryBackgroundResults of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects.MethodsFOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762.FindingsBetween Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months.InterpretationFluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function.FundingUK Stroke Association and NIHR Health Technology Assessment Programme.

Estradiol 17-beta (E2) induces epithelial proliferation, stratification, and cornification in vaginal epithelium. Our aim was to determine the respective roles of epithelial and stromal estrogen receptor-alpha (ER alpha) in these E2-induced events. Vaginal epithelium (E) and stroma (S) from adult ER alpha knockout (ko) and wild-type (wt) neonatal Balb/c mice were enzymatically separated and used to produce four types of tissue recombinants in which epithelium, stroma, or both lack functional ER alpha. Tissue recombinants were grafted into female nude mice, which were subsequently ovariectomized and treated with oil or E2. In response to E2 treatment, grafts prepared with wt-S (wt-S + wt-E and wt-S + ko-E) showed similar large increases in epithelial labeling index, indicating that E2 stimulated epithelial proliferation despite a lack of epithelial ER alpha in wt-S + ko-E tissue recombinants. Conversely, in tissue recombinants prepared with ko-S (ko-S + wt-E and ko-S + ko-E), epithelial labeling index remained at baseline levels after E2 or oil treatment, even though epithelial ER alpha were detected in ko-S + wt-E grafts. Epithelial cornification was present in wt-S + wt-E grafts from E2-treated hosts, whereas epithelium in all other tissue recombinants failed to cornify. Grafts composed of wt-S + wt-E from E2-treated hosts had highly stratified epithelium, whereas epithelial thickness was reduced almost 60% in wt-S + ko-E tissue recombinants grown in E2-treated hosts and was atrophic in all other tissue recombinants. In addition, cytokeratin 10, a marker of epithelial differentiation, was strongly expressed in wt-S + wt-E tissue recombinants grown in E2-treated hosts but was markedly reduced or absent in all other tissue recombinants. These results indicate that E2-induced vaginal epithelial proliferation is mediated indirectly through stromal ER alpha, consistent with our recent findings in uterus. Conversely, both epithelial and stromal ER alpha are required for E2-induced cornification and normal epithelial stratification. These are the first known functions attributed to epithelial ER alpha in vivo and the first time any epithelial response to E2 has been shown to involve both stromal and epithelial ER alpha.

INTRODUCTIONEstradiol-17␤ (E 2 ) stimulates uterine and vaginal epithelial proliferation in vivo [1]. E 2 also plays a critical role in other aspects of uterine and vaginal growth and adult function, and it is obligatory for normal epithelial morphogenesis, cytodifferentiation, and secretory activity in these organs. E 2 elicits its effects via the estrogen receptor (ER), which functions as a ligand-activated transcription factor to turn on target genes in E 2 -responsive tissues.For many years it was believed that E 2 acted through a single type of ER. However, the recent discovery [2, 3] of a second receptor, named ER␤ to distinguish it from the original receptor now termed ER␣, has indicated that mediation of E 2 action is more complicated than originally thought. ER␤, like ER␣, is a member of the steroid receptor superfamily, and low but detectable levels of ER␤ mRNA and protein have been detected in the rodent uterus and vagina [3][4][5]. However, E 2 treatment of ER␣ knockout (ERKO) mice does not produce characteristic estrogenic responses such as vaginal epithelial stratification and cornification, uterine epithelial DNA synthesis, or increases in uterine wet weight and mRNA levels for uterine progesterone receptor, glucose 6-phosphate dehydrogenase, or lactoferrin [6], despite the expression of ER␤ mRNA in the uterus and vagina of these animals [4]. Therefore, although the role of ER␤ in the female reproductive tract has not been established, ER␣ seems to be the critical receptor for mediating vaginal and uterine responses commonly associated with E 2 treatment. This review will therefore address only the role of ER␣ in various E 2 -induced processes such as epithelial proliferation and differentiation and will not deal with ER␤ further.ER is expressed in both epithelial and stromal cells of adult uterus and vagina [7,8], and it was initially assumed that E 2 effects on epithelium and stroma were mediated directly through ER in these tissue compartments. Several findings not consistent with this seemingly obvious mechanism of E 2 action have appeared in recent years and sug-

Summary Background Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention. Funding British Heart Foundation.