Perinatal exposure to low doses of glyphosate-based herbicide combined with a high-fat diet in adulthood causes changes in the jejunums of mice.

AIM: Exposure to pesticides and consumption of high-fat diets are widespread in society. Reports have shown that exposure to glyphosate and a high-fat diet can cause gastrointestinal disorders and increase susceptibility to obesity. Thus, this study evaluated the impacts of perinatal exposure to glyphosate followed by consumption of a high-fat diet in adulthood on the histology and morphometry of jejunums and enteric nervous system of C57BL/6 mice. MATERIAL AND METHODS: After mating, 20 C57BL/6 female mice were separated into a control group (CG) and a glyphosate group (GLY) that received water with 0.5% glyphosate. After the lactation period, some male offspring were randomly separated into CG-SD and GLY-SD (standard diet) groups or CG-HD and GLY-HD (high-fat diet) groups. After 12 weeks, jejunum samples were collected and submitted to histological analysis. KEY FINDINGS: Indirect exposure to glyphosate changed the morphometry of the intestinal wall, increased the proportion of intraepithelial lymphocytes (IELs) and goblet cells, and altered the area occupied by collagen fibers. The hyperlipidemic diet hypertrophied the jejunal total wall, total muscular and submucosal layers, decreased IELs, and increased the proportion of goblet cells. GLY-HD mice had shallower crypts, shorter villi, and less goblet cells and IELs than mice from GLY-SD group. GLY-HD also showed an increased number of neurons in myenteric and submucosal plexuses. Groups exposed to glyphosate and/or fed a high-fat diet had atrophied submucosal neurons. SIGNIFICANCE: This study suggests that perinatal glyphosate exposure combined with a high-fat diet in adulthood increases the risk of jejunum inflammation and dysfunction.

Conventional progesterone receptors (PR) B and PRA are expressed by human chondrocytes and can be involved in the pathogenesis of osteoarthritis.

To evaluate the expression, location and role of progesterone receptors (PRs) A and B in human chondrocytic cell lines, Western blotting, real time PCR analyses, transmission electron microscopy and immunogold assays were performed. By transfection and co-transfection assays, the influence of progesterone (OHPg) on estrogen receptor alpha (ERα) promoter activity was investigated. MTT and pAKT documented OHPg effects on chondrocytes survival. The PR-B and PR-A were both observed in human chondrocytes. The PR-B was evidenced both in the nucleus and in the cytosol of the cells. OHPg, through PR-B, induced ERα expression by acting at the ER promoter level affecting chondrocytes survival. We reported for the first time the expression of PRs in human chondrocytes. Interestingly, we described a novel mechanism via progesterone induction of ERα, which may explain, at least in part, the dramatic rise in OA prevalence among postmenopausal women.

Activated-farnesoid X receptor (FXR) expressed in human sperm alters its fertilising ability.

The farnesoid X receptor alpha (FXR) is a bile acid sensor activated by binding to endogenous bile acids including chenodeoxycholic acid (CDCA). Although, FXR is expressed in male reproductive tissue, the relevance of the receptor on reproduction is scarcely known. Here, we demonstrated the FXR presence and its action on several human sperm features. Western blot and immunofluorescence assays evidenced the FXR expression in human spermatozoa and the localisation in the middle piece. CDCA increasing concentrations and GW4064, synthetic ligand of FXR, were used to study the FXR influence on sperm motility, survival, capacitation, acrosome reaction and on glucose as well as lipid metabolism. Interestingly, our data showed that increasing concentrations of CDCA negatively affected sperm parameters, while the receptor blockage by (Z)-Guggulsterone and by the anti-FXR Ab reversed the effects. Intriguingly, elevated CDCA levels increased triglyceride content, while lipase and G6PDH activities were reduced with respect to untreated samples, thus impeding the metabolic reprogramming typical of the capacitated sperm. In conclusion, in this study, we demonstrated for the first time a novel target for FXR and that the activated receptor alters the acquisition of sperm fertilising ability. We showed that sperm itself express the FXR and it is responsive to specific ligands of the receptor; therefore, bile acids influence this cell both in male and in female genital tracts. It might be hypothesized that bile acid levels could be involved in infertility with idiopathic origin as these compounds are not systematically measured in men undergoing medically assisted procreation.

Mutations in the estrogen receptor alpha hormone binding domain promote stem cell phenotype through notch activation in breast cancer cell lines.

The detection of recurrent mutations affecting the hormone binding domain (HBD) of estrogen receptor alpha (ERα/ESR1) in endocrine therapy-resistant and metastatic breast cancers has prompted interest in functional characterization of these genetic alterations. Here, we explored the role of HBD-ESR1 mutations in influencing the behavior of breast cancer stem cells (BCSCs), using various BC cell lines stably expressing wild-type or mutant (Y537 N, Y537S, D538G) ERα. Compared to WT-ERα clones, mutant cells showed increased CD44+/CD24- ratio, mRNA levels of stemness genes, Mammosphere Forming Efficiency (MFE), Self-Renewal and migratory capabilities. Mutant clones exhibited high expression of NOTCH receptors/ligands/target genes and blockade of NOTCH signaling reduced MFE and migratory potential. Mutant BCSC activity was dependent on ERα phosphorylation at serine 118, since its inhibition decreased MFE and NOTCH4 activation only in mutant cells. Collectively, we demonstrate that the expression of HBD-ESR1 mutations may drive BC cells to acquire stem cell traits through ER/NOTCH4 interplay. We propose the early detection of HBD-ESR1 mutations as a challenge in precision medicine strategy, suggesting the development of tailored-approaches (i.e. NOTCH inhibitors) to prevent disease development and metastatic spread in BC mutant-positive patients.

Conditional expression of Ki-RasG12V in the mammary epithelium of transgenic mice induces estrogen receptor alpha (ERα)-positive adenocarcinoma.

Appropriate 'in vivo' models are crucial for studying breast cancer biology and evaluating the efficacy of therapeutic agents. Thus we engineered a novel transgenic mouse line expressing the human Ki-Ras bearing an activating mutation (Ki-Ras(G12V)) selectively in the mammary epithelium after lactation. These mice develop invasive ductal adenocarcinomas with 100% incidence within 3-9 months after Ki-Ras(G12V) induction. Immunophenotyping revealed that the mammary tumors express luminal markers, are positive for estrogen and progesterone receptors, negative for HER2 and have a low proliferation index. Moreover, cell lines derived from such tumors are estrogen-responsive and, when transplanted into nude mice, form tumors that respond to the antiestrogen ICI 182780. In conclusion, the mammary tumors of these transgenic mice and the derived cell lines exhibit key features of the major form of human breast cancer, that is, luminal A subtype and thus have a high potential for breast cancer research and treatment.

PTEN ELEVATION, AUTOPHAGY AND METABOLIC REPROGRAMMING MAY BE INDUCED IN HUMAN CHONDROCYTES DURING STEROIDS OR NUTRIENT DEPLETION AND OSTEOARTHRITIS.

The exact mechanisms controlling the development and progression of osteoarthritis have not yet been clarified. Our aim was to investigate new pathomechanisms, with an emphasis on novel molecular targets that might regulate human chondrocytes in osteoarthritis. As a model for studying cell survival and metabolism, C-28/I2 and T/C-28a4 human chondrocytes were grown in complete medium, in dex-tran-coated charcoal treated medium and in serum-free medium. Healthy and osteoarthritic human cartilage samples were obtained from discarded surgical material. Cell survival, PTEN, AKT, Beclin1, AMBRA, AMPK and glucose/triglyceride metabolism were evaluated by immunoblotting and spectro-photometric assays. Starvation and steroids depletion decreased cell survival concomitantly with PTEN elevation, repression of the PI3K/AKT signaling axis and autophagy activation. These experimental conditions promoted the accumulation of glucose, decreased levels of G6PDH and resulted in differen-tial expression of OXPHOS complexes. Furthermore, they induced the expression of AMPK, reduced triglyceride levels and increased lipase activity, which was accompanied by a change in chondrocytes toward a fibroblast-like morphology. In osteoarthritic human cartilage, increased PTEN, AMPK and autophagy reflected the chondrocyte responses observed during starvation and steroids depletion. In conclusion, we defined the metabolic phenotype of human chondrocytes, in which both starvation and steroids depletion induce the activation of PTEN, AMPK and autophagy signaling, concomitant with metabolic reprogramming. Our data may aid in the development of novel in vitro models for the discovery and design of drugs or nutraceuticals capable of ameliorating the course of osteoarthritis.

Farnesoid X receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through downregulation of HER2 expression.

Tamoxifen (Tam) treatment is a first-line endocrine therapy for estrogen receptor-α-positive breast cancer patients. Unfortunately, resistance frequently occurs and is often related with overexpression of the membrane tyrosine kinase receptor HER2. This is the rationale behind combined treatments with endocrine therapy and novel inhibitors that reduce HER2 expression and signaling and thus inhibit Tam-resistant breast cancer cell growth. In this study, we show that activation of farnesoid X receptor (FXR), by the primary bile acid chenodeoxycholic acid (CDCA) or the synthetic agonist GW4064, inhibited growth of Tam-resistant breast cancer cells (termed MCF-7 TR1), which was used as an in vitro model of acquired Tam resistance. Our results demonstrate that CDCA treatment significantly reduced both anchorage-dependent and anchorage-independent epidermal growth factor (EGF)-induced growth in MCF-7 TR1 cells. Furthermore, results from western blot analysis and real-time reverse transcription-PCR revealed that CDCA treatment reduced HER2 expression and inhibited EGF-mediated HER2 and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation in these Tam-resistant breast cancer cells. Transient transfection experiments, using a vector containing the human HER2 promoter region, showed that CDCA treatment downregulated basal HER2 promoter activity. This occurred through an inhibition of nuclear factor-κB transcription factor binding to its specific responsive element located in the HER2 promoter region as revealed by mutagenesis studies, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Collectively, these data suggest that FXR ligand-dependent activity, blocking HER2/MAPK signaling, may overcome anti-estrogen resistance in human breast cancer cells and could represent a new therapeutic tool to treat breast cancer patients that develop resistance.

Conventional progesterone receptors (PR) B and PRA are expressed in human spermatozoa and may be involved in the pathophysiology of varicocoele: a role for progesterone in metabolism.

The physiological roles of intracellular progesterone (PRG) receptors (PRs) have been studied intensively in female mammals, while their functions in male are scarce. Conventional PRs were evidenced in our study by Western blotting, concomitantly in healthy spermatozoa and in oligoasthenoteratozoospermic samples without and with varicocoele. Transmission electron microscopy revealed the presence of the PRs on the membrane as well as in the nucleus, mitochondria and flagellum. A reduced expression of the PRs was observed only in varicocoele spermatozoa. Responses to PRG treatment on cholesterol efflux, tyrosine phosphorylation, src and Akt activities, acrosin activity and acrosome reaction in varicocoele spermatozoa were reduced or absent. To further investigate PRG significance in human male gamete, we focused its action on lipid and glucose metabolism. The evaluation of the triglycerides content, lipase and acyl-CoA dehydrogenase activities suggests that PRG through the PRs exerts a lipolytic effect on human spermatozoa. An increase in glucose-6-phosphate dehydrogenase activity was also obtained, evidencing a role for PRG on glucose metabolism. In 'varicocoele' spermatozoa, the PRG did not induce energy consumption. The action of PRs on sperm metabolism is a novel finding that renews the importance of PRG in male fertility. Our results showed that varicocoele may lead to male factor infertility by a mechanism involving a decreased PR expression in human spermatozoa that evidences a detrimental effect on spermatozoa at the molecular level, going beyond the abnormal sperm morphology described to date.

[Diagnostic imaging in hypertrophic pyloric stenosis]. / La diagnostica per immagini nella stenosi ipertrofica del piloro.

This report discusses hypertrophic pyloric stenosis (HPS) and the current approach to diagnostic imaging in the vomiting infant. Signs and symptoms include dehydration and vigorous gastric peristalsis with vomitus. Palpation of an olive-shaped firm muscular tumor is pathognomonic of this condition. The radiographic signs of HPS are well known. Previously published criteria for the sonographic diagnosis of HPS are discussed, these include: measurements of pyloric length, diameter and muscle thickness. The thickened muscle is the most discriminated and accurate one. It was concluded that real-time ultrasound is a simple, and reliable method for the diagnosis of HPS and should be the initial imaging procedure.