Estrogen increases calcitonin gene-related peptide-immunoreactive sensory innervation of rat mammary gland

Estrogen plays important roles in preparing mammary tissue for lactation. However, estrogen also influences innervation in some tissues. We examined the effect of estrogen on peripheral innervation of mammary tissues of ovariectomized adult virgin female rats. Seven days after ovariectomy, 17beta-estradiol or placebo pellets were implanted subcutaneously, and tissues were harvested 1 week later. Estrogen treatment decreased mammary gland mass and adipocyte content, while ductal content increased and vascular composition was unaffected. Estrogen increased total areas occupied by nerves in mammary gland sections immunostained for the pan-neuronal marker protein gene product 9.5, and this increase persisted after normalizing for treatment-induced differences in gland mass. Although a significant increase in tyrosine hydroxylase-immunoreactive sympathetic nerve area was observed, no difference was detected following correction for differences in gland size, implying a conserved number of sympathetic nerves in the face of reduced gland volume. Calcitonin gene-related peptide-immunoreactive sensory nerve sectional area was also increased, and corrected nerve area remained 88% greater, indicating nerve proliferation during estrogen treatment. Total, sensory, and sympathetic innervation of the nipple and adjacent dermal tissue were unaffected by estrogen. We conclude that chronic estrogen elevation induces selective proliferation of rat mammary gland calcitonin gene-related peptide-containing nerves, which are associated primarily with blood vessels and are probably nociceptors. Because they are likely to subserve a vasodilatory function, increased innervation may promote increased blood flow necessary for milk formation during suckling. Moreover, these findings may help explain abundant anecdotal reports of increased breast sensitivity in humans under high estrogen conditions.     

17‐β‐Estradiol increases macrophage activity through activation of the G‐protein‐coupled estrogen receptor and improves the response of female mice to Cryptococcus gattii

Cryptococcus gattii (Cg) is one of the agents of cryptococcosis, a severe systemic mycosis with a higher prevalence in men than women, but the influence of the female sex hormone, 17‐β‐estradiol (E2), on cryptococcosis remains unclear. Our study shows that female mice presented delayed mortality, increased neutrophil recruitment in bronchoalveolar lavage fluid, and reduced fungal load after 24 hr of infection compared to male and ovariectomised female mice (OVX). E2 replacement restored OVX female survival. Female macrophages have more efficient fungicidal activity, which was increased by E2 and reversed by the antagonist of G‐protein‐coupled oestrogen receptor (GPER), which negatively modulates PI3K activation. Furthermore, E2 induces a reduction in Cg cell diameter, cell charge, and antioxidant peroxidase activity. In conclusion, female mice present improved control of Cg infection, and GPER is important for E2 modulation of the female response.     

Lactation‐associated infertility in Japanese monkeys (Macaca fuscata) during the breeding season

To investigate the endocrine factors in Japanese monkeys (Macaca fuscata) responsible for the suppression of the estrous cycle during the first reproductive season after delivery (150–360 days postpartum), peripheral blood was taken to measure plasma concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone, estradiol‐17β, immunoreactive (ir)‐inhibin, and cortisol. The results demonstrated that during the breeding season of lactating Japanese monkeys, circulating concentrations of FSH (1.7–2.7 ng/ml), LH (308.5–461.0 pg/ml), estradiol‐17β (<62.6 pg/ml), and progesterone (145.0–453.0 pg/ml) remained low and were similar to the nadir levels observed during both the normal menstrual cycles and the nonbreeding season. Concentrations of ir‐inhibin, which is secreted from both follicles and corpus luteum in female Japanese monkeys, were also low (300.5–585.0 pg/ml). This strongly suggests that no follicular development occurs during lactation. Serum concentrations of cortisol (261.0–519 ng/ml) were higher during lactation than during the nonbreeding season. Since babies were often seen suckling their mothers during the study, the results indicate that the increased cortisol levels were associated with suckling‐induced secretion of corticotrophin‐releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). The results of this study indicate that a long period of postpartum infertility in lactating Japanese monkeys, with apparent inhibition of follicle growth and anovulation, is due to weak gonadotropin stimulation, which may occur as the result of a suckling stimulus. Zoo Biol 22:65–76, 2003. © 2003 Wiley‐Liss, Inc.     

Estrogen metabolism in the Asian elephant (Elephas maximus)

Estradiol-17β metabolism was studied in two female Asian elephants (Elephas maximus). In an initial study, 500 μCi of tritiated estradiol-17β was injected iv into a single animal, and 0, 30 and 60 min serum samples were collected as well as all excreted urine and feces for 24 hr. In a second study, 1.5 mg unlabeled estradiol-17β was injected iv into a second animal and 0, 5, 15, 30, and 60 min serum samples and a 30 min urine sample were collected postinjection. Analyses of samples from both studies demonstrated a rapid conversion of free estradiol to conjugated forms in the serum. The first (5 min) serum sample following the injection of unlabelled estradiol contained unconjugated estradiol: conjugated estradiol: conjugated estrone at a ratio of 60: 29: 10, respectively, and at 30 min a ratio of 33: 43: 24. The urinary estrogen metabolites were in the conjugated form with an estradiol: estrone ratio of 60: 40. No radiolabeled estrogen was found in the fecal samples during the 24 hr following administration of the radiolabeled estradiol. These data indicate a rapid clearance of circulating free estradiol in the elephant, with a major metabolite in the serum and urine being estradiol conjugate. © 1992 Wiley-Liss, Inc.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

17β‐Estradiol Protects Human Eyelid‐Derived Adipose Stem Cells against Cytotoxicity and Increases Transplanted Cell Survival in Spinal Cord injury

Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b‐estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose‐derived stem cells (hEASCs) in vitro with hydrogen peroxide (H₂O₂)‐induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H₂O₂‐induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

A dimer model of human calcitonin13‐32 forms an α‐helical structure and robustly aggregates in 50% aqueous 2,2,2‐trifluoroethanol solution

Determining the cause of human calcitonin (hCT) aggregation could be of help in the effort to utilize hCT for treatment of hypercalcemia. Here we report that a dimer model of hCT13‐32 aggregated to a greater degree than native hCT under aqueous 2,2,2‐trifluoroethanol conditions. Analyses using circular dichroism spectroscopy, thioflavine‐T binding assays and atomic force microscopy suggest that the α‐helical portion of hCT is important for initiation of the aggregation process, which yields long fibrils. Dimerization, which stabilizes the β‐sheet structure of hCT, enhances aggregation potency. Dimerization of hCT stabilizes the α‐helix under aqueous TFE conditions, leading to the long fibril formation. Up to now, there have been no reports of using a dimer model to investigate the properties of hCT aggregation. Our findings could potentially serve as the basis for development of novel hCT derivatives that could be utilized for treatment of hypercalcemia, as well as for development of novel therapeutics for other ailments caused by amyloid peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Suppression of IL‐8‐Src signalling axis by 17β‐estradiol inhibits human mesenchymal stem cells‐mediated gastric cancer invasion

Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs‐mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin‐8 (IL‐8) protein. Administration of IL‐8 specific neutralizing antibody significantly inhibits HBMMSCs‐mediated gastric cancer motility. Treatment of recombinant IL‐8 soluble protein confirmed the role of IL‐8 in mediating HBMMSCs‐up‐regulated cell motility. IL‐8 up‐regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17β ‐estradiol inhibit HBMMSCS‐induced cell motility via suppressing activation of IL8‐Src signalling in human gastric cancer cells. 17β‐estradiol inhibits IL8‐up‐regulated Src downstream target proteins including p‐Cas, p‐paxillin, p‐ERK1/2, p‐JNK1/2, MMP9, tPA and uPA. These results suggest that 17β‐estradiol significantly inhibits HBMMSCS‐induced invasive motility through suppressing IL8‐Src signalling axis in human gastric cancer cells.     

17‐β estradiol attenuates the pro‐oxidant activity of corticotropin‐releasing hormone in macroendothelial cells

Corticotropin‐releasing hormone, which is the predominant regulator of neuroendocrine responses to stress, attenuates inflammation through stimulation of glucocorticoid release. Enhanced corticotropin‐releasing hormone expression has been detected in inflammatory cells of the vascular endothelium, where it acts as a local regulator of endothelial redox homeostasis. Estrogens have beneficial effects on endothelial integrity and function, though the mechanism underlying their antioxidative effect remains as yet largely unknown. We therefore investigated the effect of 17β‐estradiol on pro‐oxidant action of corticotropin‐releasing hormone in vitro in macroendothelial cells, and, more specifically, the role of 17β‐estradiol on corticotropin‐releasing hormone‐induced activities/release of the antioxidant enzymes namely, endothelial nitric oxide synthase, superoxide dismutase, catalase, and glutathione. We observed that 17β‐estradiol abolished the stimulatory effect of corticotropin‐releasing hormone on intracellular reactive oxygen species levels and counteracted its inhibitory effect on endothelial nitric oxide synthase activity and nitric oxide release. In addition, 17β‐estradiol significantly induced superoxide dismutase and catalase activity, an effect that was not significantly influenced by corticotropin‐releasing hormone. Finally, 17β‐estradiol significantly increased glutathione levels and the glutathione/glutathione + glutathione disulfide ratio, an action that was partially blocked by corticotropin‐releasing hormone. Our results reveal that 17β‐estradiol counterbalances corticotropin‐releasing hormone‐mediated pro‐inflammatory action and thereby maintains the physiological threshold of the endothelial cell redox environment. These observations may be of importance, considering the protective role of estrogen in the development of atherosclerosis.