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Breast cancer resistance protein (BCRP) is known for its protective function against the toxic effects of exogenous compounds. In addition to this, a role in the transport of endogenous compounds has been described. Since BCRP in the plasma membrane was shown to be regulated by sex steroids, we investigated the presence and possible role of BCRP in steroid hormone-producing organs. Therefore, the presence and localization of Bcrp was investigated in endocrine organs of wild-type mice. Furthermore, the interaction of various steroid hormones with human BCRP activity was studied. Quantitative PCR revealed Bcrp mRNA in the pituitary and adrenal glands, pancreas, ovary, testis and adipose tissue. Immunohistochemistry revealed the presence of Bcrp in the cortex of the adrenal gland and in plasma membranes of adipocytes. In the pituitary gland, pancreas, ovary and testis, Bcrp was mainly located in the capillaries. The interaction between BCRP and 12 steroid hormones was studied using membrane vesicles of HEK293-BCRP cells. Estradiol, testosterone, progesterone and androstenedione inhibited BCRP-mediated uptake of 3H-estrone sulphate (E1S) most potently, with calculated inhibitory constant (Ki) values of 5.0 ± 0.2, 36 ± 14, 14.7 ± 1.3 and 217 ± 13 μM, respectively. BCRP function was attenuated non-competitively, which implies an allosteric inhibition of BCRP-mediated E1S transport by these steroids. In conclusion, localization of Bcrp in endocrine organs together with the efficient allosteric inhibition of the efflux pump by steroid hormones are suggestive for a role for BCRP in steroid hormone regulation.

Tribbles homolog 3 (TRIB3) is a scaffold protein activated under hypoxic conditions and involved in several cell survival and proliferation pathways. Recently, we reported opposite associations of TRIB3 mRNA and protein with breast cancer prognosis. In this study, we investigated this discrepancy between TRIB3 mRNA and protein in human breast cancer. We provide several lines of evidence demonstrating that TRIB3 is a stabile protein which levels are not controlled by rapid protein breakdown. Interestingly, we were able to show that during anoxia TRIB3 mRNA translation was profoundly inhibited. Hypoxia induced micro RNA 24 was not responsible for the translational repression of TRIB3. Furthermore miRNA-24 expression levels in breast cancer patient specimens showed no correlation with TRIB3 mRNA or TRIB3 protein levels, or with prognosis. Thus, the expression of miRNA-24 does not explain the difference between mRNA and protein expression of TRIB3 in this cohort of breast cancer patients. In conclusion, TRIB3 protein is a stable protein which levels are predominantly regulated by translational control of TRIB3 mRNA transcript.

Background.Identification of new therapeutic targets remains an imperative goal to improve the morbidity and mortality associated with severe sepsis and septic shock. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, has recently emerged as a critical mediator of innate immunity and experimental sepsis, and it is an attractive new target for the treatment of sepsis. Methods.Circulating concentrations of MIF were measured in 2 clinical trial cohorts of 145 pediatric and adult patients who had severe sepsis or septic shock caused predominantly by infection with Neisseria meningitidis or other gram-negative bacteria, to study the kinetics of MIF during sepsis, to analyze the interplay between MIF and other mediators of sepsis or stress hormones (adrenocorticotropic hormone and cortisol), and to determine whether MIF is associated with patient outcome. Results.Circulating concentrations of MIF were markedly elevated in 96% of children and adults who had severe sepsis or septic shock, and they remained elevated for several days. MIF levels were correlated with sepsis severity scores, presence of shock, disseminated intravascular coagulation, urine output, blood pH, and lactate and cytokine levels. High levels of MIF were associated with a rapidly fatal outcome. Moreover, in meningococcal sepsis, concentrations of MIF were positively correlated with adrenocorticotropic hormone levels and negatively correlated with cortisol levels and the cortisol : adrenocorticotropic hormone ratio, suggesting an inappropriate adrenal response to sepsis. Conclusions.MIF is markedly and persistently up-regulated in children and adults with gram-negative sepsis and is associated with parameters of disease severity, with dysregulated pituitary-adrenal function in meningococcal sepsis, and with early death.

Introduction Treatment with antibodies directed against programed‐cell death ligand 1 (PD‐L1) is a novel therapy for patients with gestational trophoblastic disease. Assessment of PD‐L1 expression in tumor tissue is commonly used to identify patients who might benefit from anti‐PD‐L1 treatment. Multiple antibodies are available to detect PD‐L1‐expressing cells, and percentages of PD‐L1‐expressing cells in samples of patients with gestational trophoblastic disease indicated by these antibodies differ substantially. This raises the question which PD‐L1 antibody best reflects PD‐L1 expression to select patients for treatment. Material and methods Seven commercially available antibodies for PD‐L1 staining (E1L3N, 73–10, 22C3, CAL10, SP142, 28–8, SP263) were validated on Chinese hamster ovarian (CHO) cells transfected with PD‐L1, PD‐L2, wildtype CHO cells and tonsil tissue. Next, four complete hydatidiform moles and four choriocarcinomas were stained. Samples were independently assessed by two pathologists. Results All seven antibodies showed membranous staining in the PD‐L1‐transfected CHO cells. E1L3N and 22C3 scored the highest percentages of PD‐L1‐positive cells (70%–90% and 60%–70%, respectively). E1L3N stained the cytoplasm of non‐transfected CHO cells and was excluded from analysis. The remaining six antibodies predominantly stained syncytiotrophoblast cells of both complete hydatidiform moles and choriocarcinomas. The percentage of PD‐L1‐stained trophoblast cells and staining intensity varied substantially per used PD‐L1 antibody and between complete hydatidiform moles and choriocarcinomas. Agreement between pathologists was best with 22C3 (intraclass correlation coefficient 0.94–0.96). Conclusions Based on staining results of the CHO cells, gestational trophoblastic disease samples and intraclass correlation coefficient, 22C3 seems the most suitable for adequate detection of PD‐L1‐expressing trophoblast cells. All antibodies detected PD‐L1‐expressing cells in the gestational trophoblastic disease samples, though with great variability, hampering comparison of results between studies in this rare disease and emphasizing the need for uniformity in detecting PD‐L1‐expressing cells.

Abstract This review provides the reader with current insights on testicular adrenal rest tumors (TARTs), a complication in male patients with congenital adrenal hyperplasia (CAH). In recent studies, an overall TART prevalence of 40% (range, 14% to 89%) in classic patients with CAH is found. Reported differences are mainly caused by the method of detection and the selected patient population. Biochemically, histologically, and molecularly, TARTs exhibit particular adrenal characteristics and were therefore thought to originate from aberrant adrenal cells. More recently, TARTs have been found to also exhibit testicular characteristics. This has led to the hypothesis of pluripotent cells as the origin of TARTs. High concentrations of ACTH could cause hyperplasia of these pluripotent cells, as TARTs appear to be associated with poor hormonal control with concomitant elevated ACTH. Unfortunately, as yet there are no methods to prevent the development of TARTs, nor are there guidelines to treat patients with TARTs. Intensified glucocorticoid treatment could improve fertility status in some cases, although studies report contradicting results. TARTs can also lead to irreversible testicular damage, and therefore semen cryopreservation could be offered to patients with TARTs. Further research should focus on the etiology and pharmacological treatment to prevent TART development or to treat TARTs and improve the fertility status of patients with TARTs.

Excessive or persistent proinflammatory cytokine production plays a central role in autoimmune diseases. Acute activation of the sympathetic nervous system attenuates the innate immune response. However, both the autonomic nervous system and innate immune system are regarded as systems that cannot be voluntarily influenced. Herein, we evaluated the effects of a training program on the autonomic nervous system and innate immune response. Healthy volunteers were randomized to either the intervention (n = 12) or control group (n = 12). Subjects in the intervention group were trained for 10 d in meditation (third eye meditation), breathing techniques (i.a., cyclic hyperventilation followed by breath retention), and exposure to cold (i.a., immersions in ice cold water). The control group was not trained. Subsequently, all subjects underwent experimental endotoxemia (i.v. administration of 2 ng/kg Escherichia coli endotoxin). In the intervention group, practicing the learned techniques resulted in intermittent respiratory alkalosis and hypoxia resulting in profoundly increased plasma epinephrine levels. In the intervention group, plasma levels of the anti-inflammatory cytokine IL-10 increased more rapidly after endotoxin administration, correlated strongly with preceding epinephrine levels, and were higher. Levels of proinflammatory mediators TNF-α, IL-6, and IL-8 were lower in the intervention group and correlated negatively with IL-10 levels. Finally, flu-like symptoms were lower in the intervention group. In conclusion, we demonstrate that voluntary activation of the sympathetic nervous system results in epinephrine release and subsequent suppression of the innate immune response in humans in vivo. These results could have important implications for the treatment of conditions associated with excessive or persistent inflammation, such as autoimmune diseases.

In a previous study, we detected a significant association between phosphoserine aminotransferase 1 (PSAT1) hyper-methylation and mRNA levels to outcome to tamoxifen treatment in recurrent disease. We here aimed to study the association of PSAT1 protein levels to outcome upon tamoxifen treatment and to obtain more insight in its role in tamoxifen resistance. A cohort of ER positive, hormonal therapy naïve primary breast carcinomas was immunohistochemically (IHC) stained for PSAT1. Staining was analyzed for association with patient’s time to progression (TTP) and overall response on first-line tamoxifen for recurrent disease. PSAT1 mRNA levels were also assessed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR; n = 161) and Affymetrix GeneChip (n = 155). Association of PSAT1 to biological pathways on tamoxifen outcome were assessed by global test. PSAT1 protein and mRNA levels were significantly associated to poor outcome to tamoxifen treatment. When comparing PSAT1 protein and mRNA levels, IHC and RT-qPCR data showed a significant association. Global test results showed that cytokine and JAK-STAT signaling were associated to PSAT1 expression. We hereby report that PSAT1 protein and mRNA levels measured in ER positive primary tumors are associated with poor clinical outcome to tamoxifen.

Purpose To assess the benefit from adjuvant systemic tamoxifen therapy in breast cancer risk groups identified by the previously established prognostic 76-gene signature. Methods In 300 lymph node-negative (LNN), estrogen receptor-positive (ER+) breast cancer patients (136 treated with adjuvant tamoxifen, 164 having received no systemic adjuvant therapy), distant metastasis-free survival (DMFS) as a function of the 76-gene signature was determined in a multicenter fashion. Results In 136 tamoxifen-treated patients, the 76-gene signature identified a group of patients with a poor prognosis [hazard ratio (HR), 4.62; P = 0.0248]. These patients showed a 12.3% absolute benefit of tamoxifen in 10-year DMFS (HR, 0.52; P = 0.0318) compared with untreated high-risk patients. This represented a 71% increase in relative benefit compared with the 7.2% absolute benefit observed for all 300 patients without using the gene signature. In the low-risk group there was no significant 10-year DMFS benefit of tamoxifen. Conclusions The 76-gene signature defines high-risk patients who benefit from adjuvant tamoxifen therapy. Although we did not study the value of chemotherapy in this study, low-risk patients identified by the 76-gene signature have a prognosis good enough that chemotherapy would be difficult to justify. The prognosis of these patients is sufficiently good, in fact, that a disease-free benefit for tamoxifen therapy is difficult to prove, though benefits in terms of loco-regional relapse and a reduction in risk for contralateral breast cancer might justify hormonal therapy in these patients.

Objectives Before diffusion-weighted imaging (DWI) can be implemented in standard clinical practice for response monitoring, data on reproducibility are needed to assess which differences outside the range of normal variation can be detected in an individual patient. In this study we assessed the reproducibility of the apparent diffusion coefficient (ADC) values in colorectal liver metastases. To provide a biological basis for these values, their relation with histopathology was assessed. Methods DWI was performed twice in 1 week in patients scheduled for metastasectomy of colorectal liver metastases. Correlation between ADC values and apoptosis marker p53, anti-apoptotic protein BCL-2, proliferation marker Ki67 and serum vascular endothelial growth factor (VEGF) concentration were assessed. Results A good reproducibility coefficient of the mean ADC (coefficient of reproducibility 0.20 × 10 −3  mm 2 /s) was observed in colorectal liver metastases ( n  = 21). The ADC value was related to the proliferation index and BCL-2 expression of the metastases. Furthermore, in metastases recently treated with systemic therapy, the ADC was significantly higher (1.27 × 10 −3  mm 2 /s vs 1.05 × 10 −3  mm 2 /s, P  = 0.02). Conclusions The good reproducibility, correlation with histopathology and implied sensitivity for systemic treatment-induced anti-tumour effects suggest that DWI might be an excellent tool to monitor response in metastatic colorectal cancer. Key Points • ADC values are becoming important oncological biomarkers • DWI provides reproducibile ADC values in colorectal liver metastases • The coefficient of reproducibility of the mean ADC is 0.20 × 10 −3   mm 2 /s • ADC values correlate with proliferation index and are related to BCL-2 expression

Water drinking acutely increases sympathetic activity in human subjects. In animals, the response appears to be mediated through transient receptor potential channel TRPV4 activation on osmosensitive hepatic spinal afferents, described as osmopressor response. We hypothesized that hepatic denervation attenuates water drinking-induced sympathetic activation. We studied 20 liver transplant recipients (44±2.6 years, 1.2±0.1 years post transplant) as model of hepatic denervation and 20 kidney transplant recipients (43±2.6 years, 0.8±0.1 years post transplant) as immunosuppressive drug matched control group. Before and after 500 ml water ingestion, we obtained venous blood samples for catecholamine analysis. We also monitored brachial and finger blood pressure, ECG, and thoracic bioimpedance. Plasma norepinephrine concentration had changed by 0.01±0.07 nmol/l in liver and by 0.21±0.07 nmol/l in kidney transplant recipients (p<0.05 between groups) after 30-40 minutes of water drinking. While blood pressure and systemic vascular resistance increased in both groups, the responses tended to be attenuated in liver transplant recipients. Our findings support the idea that osmosensitive hepatic afferents are involved in water drinking-induced sympathetic activation in human subjects. ClinicalTrials.gov NCT01237431.