In Vivo Imaging of Activated Estrogen Receptors in Utero by Estrogens and Bisphenol A

Environmental estrogens are of particular concern when exposure occurs during embryonic development. Although there are good models to study estrogenic activity of chemicals in adult animals, developmental exposure is much more difficult to test. The weak estrogenic activity of the environmental estrogen bisphenol A (BPA) in embryos is controversial. We have recently generated transgenic mice that carry a reporter construct with estrogen-responsive elements coupled to luciferase. We show that, using this in vivo model in combination with the IVIS imaging system, activation of estrogen receptors (ERs) by maternally applied BPA and other estrogens can be detected in living embryos in utero. Eight hours after exposure to 1 mg/kg BPA, ER transactivation could be significantly induced in the embryos. This was more potent than would be estimated from in vitro assays, although its intrinsic activity is still lower than that of diethylstilbestrol and 17β-estradiol dipropionate. On the basis of these results, we conclude that the estrogenic potency of BPA estimated using in vitro assays might underestimate its estrogenic potential in embryos

Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities

Identification of nuclear receptor-mediated endocrine activities is important in a variety of fields, ranging from pharmacological and clinical screening, to food and feed safety, toxicological monitoring, and risk assessment. Traditionally animal studies such as the Hershberger and Allen-Doisy tests are used for the assessment of androgenic and estrogenic potencies, respectively. To allow fast analysis of the activities of new chemicals, food additives, and pharmaceutical compounds, high-throughput screening strategies have been developed. Here, a panel of mainly steroidal compounds, screened in different in vitro assays, was compared with two human U2-OS cell line-based CALUX® (Chemically Activated LUciferase eXpression) reporter gene assays for androgens (AR CALUX) and estrogens (ER CALUX). Correlations found between the data of these two CALUX reporter gene assays and data obtained with other in vitro screening assays measuring receptor binding or reporter gene activation (CHO cell line-based) were good (correlation coefficients (

Outcome in hip fracture patients related to anemia at admission and allogeneic blood transfusion: an analysis of 1262 surgically treated patients

Background: Anemia is more often seen in older patients. As the mean age of hip fracture patients is rising, anemia is common in this population. Allogeneic blood transfusion (ABT) and anemia have been pointed out as possible risk factors for poorer outcome in hip fracture patients. Methods. In the timeframe 2005-2010, 1262 admissions for surgical treatment of a hip fracture in patients aged 65 years and older were recorded. Registration was prospective from 2008 on. Anemic and non-anemic patients (based on hemoglobin level at admission) were compared regarding clinical characteristics, mortality, delirium incidence, LOS, discharge to a nursing home and the 90-day readmission rate. Receiving an ABT, age, gender, ASA classification, type of fracture and anesthesia were used as possible confounders in multivariable regression analysis. Results: The prevalence of anemia and the rate of ABT both were 42.5%. Anemic patients were more likely to be older and men and had more often a trochanteric fracture, a higher ASA score and received more often an ABT. In univariate analysis, the 3- and 12-month mortality rate, delirium incidence and discharge to a nursing home rate were significantly worse in preoperatively anemic patients. In multivariable regression analysis, anemia at admission was a significant risk factor for discharge to a nursing home and readmission < 90 days, but not for mortality. Indication for ABT, age and ASA classification were independent risk factors for mortality at all moments, only the mortality rate for the 3-12 month interval was not influenced by ABT. An indication for an ABT was the largest negative contributor to a longer LOS (OR 2.26, 95% CI 1.73-2.94) and the second largest for delirium (OR 1.67, 95% CI 1.28-2.20). Conclusions: This study has demonstrated that anemia at admission and postoperative anemia needing an ABT (PANT) were independent risk factors for worse outcome in hip fracture patients. In multivariable regression analysis, anemia as such had no effect on mortality, due to a rescue effect of PANT. In-hospital, 3- and 12-month mortality was negatively affected by PANT, with the main effect in the first 3 months postoperatively

iPSC-based modeling of RAG2 severe combined immunodeficiency reveals multiple T cell developmental arrests

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7(-)CD5(-) to CD4(+)CD8(+). The impaired differentiation was accompanied by an increase in CD7(-)CD56(+)CD33(+) natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks

CD40-mediated amplification of local immunity by epithelial cells is impaired by HPV

The interaction between the transmembrane glycoprotein surface receptor CD40 expressed by skin epithelial cells (ECs) and its T-cell-expressed ligand CD154 was suggested to exacerbate inflammatory skin diseases. However, the full spectrum of CD40-mediated effects by ECs underlying this observation is unknown. Therefore, changes in gene expression after CD40 ligation of ECs were studied by microarrays. CD40-mediated activation for 2 hours stimulated the expression of a coordinated network of immune-involved genes strongly interconnected by IL8 and TNF, whereas after 24 hours anti-proliferative and anti-apoptotic genes were upregulated. CD40 ligation was associated with the production of chemokines and the attraction of lymphocytes and myeloid cells from peripheral blood mononuclear cells (PBMCs). Thus, CD40-mediated activation of ECs resulted in a highly coordinated response of genes required for the local development and sustainment of adaptive immune responses. The importance of this process was confirmed by a study on the effects of human papilloma virus (HPV) infection to the EC's response to CD40 ligation. HPV infection clearly attenuated the magnitude of the response to CD40 ligation and the EC's capacity to attract PBMCs. The fact that HPV attenuates CD40 signaling in ECs indicates the importance of the CD40-CD154 immune pathway in boosting cellular immunity within epithelia

Identification of major dioxin-like compounds and androgen receptor antagonist in acid-treated tissue extracts of high trophic-level animals

We evaluated the applicability of combining in vitro bioassays with instrument analyses to identify potential endocrine disrupting pollutants in sulfuric acid-treated extracts of liver and/or blubber of high trophic-level animals. Dioxin-like and androgen receptor (AR) antagonistic activities were observed in Baikal seals, common cormorants, raccoon dogs, and finless porpoises by using a panel of rat and human cell-based chemical-activated luciferase gene expression (CALUX) reporter gene bioassays. On the other hand, no activity was detected in estrogen receptor α (ERα)-, glucocorticoid receptor (GR)-, progesterone receptor (PR)-, and peroxisome proliferator-activated receptor γ2 (PPARγ2)-CALUX assays with the sample amount applied. All individual samples (n = 66) showed dioxin-like activity, with values ranging from 21 to 5500 pg CALUX-2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (TEQ)/g-lipid. Because dioxins are expected to be strong contributors to CALUX-TEQs, the median theoretical contribution of dioxins calculated from the result of chemical analysis to the experimental CALUX-TEQs was estimated to explain up to 130% for all the tested samples (n = 54). Baikal seal extracts (n = 31), but not other extracts, induced AR antagonistic activities that were 8-150 μg CALUX-flutamide equivalent (FluEQ)/g-lipid. p,p′-DDE was identified as an important causative compound for the activity, and its median theoretical contribution to the experimental CALUX-FluEQs was 59% for the tested Baikal seal tissues (n = 25). Our results demonstrate that combining in vitro CALUX assays with instrument analysis is useful for identifying persistent organic pollutant-like compounds in the tissue of wild animals on the basis of in vitro endocrine disruption toxicity. © 2011 American Chemical Society

A Family of Water Immiscible, Dipolar Aprotic, Diamide Solvents from Succinic Acid

Three dipolar aprotic solvents were designed to possess high dipolarity and low toxicity: N , N , N ', N '-tetrabutylsuccindiamide (TBSA), N , N '-diethyl- N , N '-dibutylsuccindiamide (EBSA), N , N '-dimethyl- N , N '-dibutylsuccindiamide (MBSA). They were synthesized catalytically using a K60 silica catalyst in a solventless system. Their water-immiscibility stands out as an unusual and useful property for dipolar aprotic solvents. They were tested in a model Heck reaction, metal-organic framework syntheses, and a selection of polymer solubility experiments where their performances were found to be comparable to traditional solvents. Furthermore, MBSA was found to be suitable for the production of an industrially-relevant membrane from polyethersulphone. An integrated approach involving in silico analysis based on available experimental information, prediction model outcomes and read across data, as well as a panel of in vitro reporter gene assays covering a broad range of toxicological endpoints was used to assess toxicity. These in silico and in vitro tests suggested no alarming indications of toxicity in the new solvents

iPSC-Based Modeling of RAG2 Severe Combined Immunodeficiency Reveals Multiple T Cell Developmental Arrests

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7−CD5− to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7−CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.In this article, Mikkers