Categorizing the characteristics of human carcinogens: a need for specificity.

The International Agency for Research on Cancer (IARC) has recently proposed employing "ten key characteristics of human carcinogens" (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

Admission cardiotocography versus intermittent auscultation of the fetal heart in low-risk pregnancy during evaluation for possible labour admission - a multicentre randomised trial: the ADCAR trial.

OBJECTIVE: To assess the effect of admission cardiotocography (ACTG) versus intermittent auscultation (IA) of the fetal heart (FH) in low-risk pregnancy during assessment for possible labour on caesarean section rates. DESIGN: A parallel multicentre randomised trial. SETTING: Three maternity units in the Republic of Ireland. POPULATION: Healthy, low-risk pregnant women, at term and ≥ 18 years old, who provided written informed consent. METHODS: Women were randomised to receive IA of the FH or 20 minutes ACTG on admission for possible labour onset, using remote telephone randomisation. Both groups received IA during labour, with conversion to continuous CTG as clinically indicated. MAIN OUTCOME MEASURES: Caesarean section (primary outcome), obstetric interventions (e.g. continuous CTG during labour, fetal blood sampling, augmentation of labour) and neonatal morbidity (e.g. metabolic acidosis, admission to the neonatal intensive care unit, neonatal death). RESULTS: Based on 3034 women (1513 and 1521 randomised to IA and ACTG, respectively), there was no statistical difference between the groups in caesarean section [130 (8.6%) and 105 (6.9%) for IA and ACTG groups, respectively; relative risk (RR) 1.24; 95% CI 0.97-1.58], or in any other outcome except for use of continuous CTG during labour, which was lower in the IA group (RR 0.90, 95% CI 0.86-0.93). CONCLUSION: Our study demonstrates no differences in obstetric or neonatal outcomes between IA and ACTG for women with possible labour onset, other than an increased risk for continuous CTG in women receiving ACTG. TWEETABLE ABSTRACT: No differences in outcomes between intermittent auscultation and admission cardiotocography for women with possible labour onset.

Health economic analysis of a cluster-randomised trial (OptiBIRTH) designed to increase rates of vaginal birth after caesarean section.

OBJECTIVE: To perform a health economic analysis of an intervention designed to increase rates of vaginal birth after caesarean, compared with usual care. DESIGN: Economic analysis alongside the cluster-randomised OptiBIRTH trial (Optimising childbirth by increasing vaginal birth after caesarean section (VBAC) through enhanced women-centred care). SETTING: Fifteen maternity units in three European countries - Germany (five), Ireland (five), and Italy (five) - with relatively low VBAC rates. POPULATION: Pregnant women with a history of one previous lower-segment caesarean section; sites were randomised (3:2) to intervention or control. METHODS: A cost-utility analysis from both societal and health-services perspectives, using a decision tree. MAIN OUTCOME MEASURES: Costs and resource use per woman and infant were compared between the control and intervention group by country, from pregnancy recognition until 3 months postpartum. Based on the caesarean section rates, and maternal and neonatal morbidities and mortality, the incremental cost-utility ratios were calculated per country. RESULTS: The mean difference in costs per quality-adjusted life years (QALYs) gained from a societal perspective between the intervention and the control group, using a probabilistic sensitivity analysis, was: €263 (95% CI €258-268) and 0.008 QALYs (95% CI 0.008-0.009 QALYs) for Germany, €456 (95% CI €448-464) and 0.052 QALYs (95% CI 0.051-0.053 QALYs) for Ireland, and €1174 (95% CI €1170-1178) and 0.006 QALYs (95% CI 0.005-0.007 QALYs) for Italy. The incremental cost-utility ratios were €33,741/QALY for Germany, €8785/QALY for Ireland, and €214,318/QALY for Italy, with a 51% probability of being cost-effective for Germany, 92% for Ireland, and 15% for Italy. CONCLUSION: The OptiBIRTH intervention was likely to be cost-effective in Ireland and Germany. TWEETABLE ABSTRACT: The OptiBIRTH intervention (to increase VBAC rates) is likely to be cost-effective in Germany and Ireland.

Dynamics of Fluorescent Imaging for Rapid Tear Thinning.

A previous mathematical model has successfully simulated the rapid tear thinning caused by glob (thicker lipid) in the lipid layer. It captured a fast spreading of polar lipid and a corresponding strong tangential flow in the aqueous layer. With the simulated strong tangential flow, we now extend the model by adding equations for conservation of solutes, for osmolarity and fluorescein, in order to study their dynamics. We then compare our computed results for the resulting intensity distribution with fluorescence experiments on the tear film. We conclude that in rapid thinning, the fluorescent intensity can linearly approximate the tear film thickness well, when the initial fluorescein concentration is small. Thus, a dilute fluorescein is recommended for visualizing the rapid tear thinning during fluorescent imaging.

Definitions, measurements and prevalence of fear of childbirth: a systematic review.

BACKGROUND: Fear of Childbirth (FOC) is a common problem affecting women's health and wellbeing, and a common reason for requesting caesarean section. The aims of this review were to summarise published research on prevalence of FOC in childbearing women and how it is defined and measured during pregnancy and postpartum, and to search for useful measures of FOC, for research as well as for clinical settings. METHODS: Five bibliographic databases in March 2015 were searched for published research on FOC, using a protocol agreed a priori. The quality of selected studies was assessed independently by pairs of authors. Prevalence data, definitions and methods of measurement were extracted independently from each included study by pairs of authors. Finally, some of the country rates were combined and compared. RESULTS: In total, 12,188 citations were identified and screened by title and abstract; 11,698 were excluded and full-text of 490 assessed for analysis. Of these, 466 were excluded leaving 24 papers included in the review, presenting prevalence of FOC from nine countries in Europe, Australia, Canada and the United States. Various definitions and measurements of FOC were used. The most frequently-used scale was the W-DEQ with various cut-off points describing moderate, severe/intense and extreme/phobic fear. Different 3-, 4-, and 5/6 point scales and visual analogue scales were also used. Country rates (as measured by seven studies using W-DEQ with ≥85 cut-off point) varied from 6.3 to 14.8%, a significant difference (chi-square = 104.44, d.f. = 6, p < 0.0001). CONCLUSIONS: Rates of severe FOC, measured in the same way, varied in different countries. Reasons why FOC might differ are unknown, and further research is necessary. Future studies on FOC should use the W-DEQ tool with a cut-off point of ≥85, or a more thoroughly tested version of the FOBS scale, or a three-point scale measurement of FOC using a single question as 'Are you afraid about the birth?' In this way, valid comparisons in research can be made. Moreover, validation of a clinical tool that is more focussed on FOC alone, and easier than the longer W-DEQ, for women to fill in and clinicians to administer, is required.

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis.

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4(+) T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.

Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus.

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.

Reproducibility in science: improving the standard for basic and preclinical research.

Medical and scientific advances are predicated on new knowledge that is robust and reliable and that serves as a solid foundation on which further advances can be built. In biomedical research, we are in the midst of a revolution with the generation of new data and scientific publications at a previously unprecedented rate. However, unfortunately, there is compelling evidence that the majority of these discoveries will not stand the test of time. To a large extent, this reproducibility crisis in basic and preclinical research may be as a result of failure to adhere to good scientific practice and the desperation to publish or perish. This is a multifaceted, multistakeholder problem. No single party is solely responsible, and no single solution will suffice. Here we review the reproducibility problems in basic and preclinical biomedical research, highlight some of the complexities, and discuss potential solutions that may help improve research quality and reproducibility.

Tribute to Donald Metcalf.

A collection of tributes and remembrances from esteemed colleagues, mentees, and friends on the life and work of "the father of hematopoietic cytokines".

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates.

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia.

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure, and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL3R) levels are a constant yet puzzling feature, as this receptor lacks tyrosine kinase activity. Here, we show that the heterodimeric IL3Rα/ßc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Rα/ßc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, in which high IL3Rα/ßc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, while low ratios mediate differentiation. Our study establishes a new paradigm in which alternative cytokine receptor stoichiometries differentially regulate cell fate, a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance. SIGNIFICANCE: Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.

Functional erythropoietin receptor is undetectable in endothelial, cardiac, neuronal, and renal cells.

Erythropoiesis stimulating agents (ESAs) have been reported to activate erythropoietin receptors (EpoR) on cell types, including endothelial, neuronal, renal tubule, and cardiac cells. ESAs have also been reported to promote angiogenesis. However, those findings are controversial and confounded by methodologic issues. We show that EpoR mRNA was detected in essentially all cell types examined, including primary human endothelial, renal, cardiac, and neuronal cells but 10- to 100-fold lower than Epo-responsive cells using quantitative reverse-transcribed polymerase chain reaction. Total endothelial EpoR protein examined using a new monoclonal antibody was low to undetectable. Surface EpoR on endothelial cells was not detected using [(125)I]-rHuEpo surface-binding studies. There was no evidence of ESA-induced intracellular signaling in endothelial cells. There was a similar lack of EpoR expression and signaling in other cell types examined. Experiments were performed examining ESA function on these cells. An in vivo rat corneal angiogenesis assay demonstrated neo-vessel formation in response to recombinant human vascular endothelial growth factor (rHuVEGF). However, recombinant mouse Epo did not induce vessel formation. Similarly, ESAs did not reproducibly provide cytoprotection to neuronal, renal, or cardiac cells. Taken together, our data challenge the notion of presence or function of EpoR on nonhematopoietic cells, and call into question the preclinical basis for clinical studies exploring direct, "pleiotropic" actions of ESAs.

Absence of functional EpoR expression in human tumor cell lines.

Certain oncology trials showed worse clinical outcomes in the erythropoiesis-stimulating agent (ESA) arm. A potential explanation was that ESA-activated erythropoietin (Epo) receptors (EpoRs) promoted tumor cell growth. Although there were supportive data from preclinical studies, those findings often used invalidated reagents and methodologies and were in conflict with other studies. Here, we further investigate the expression and function of EpoR in tumor cell lines. EpoR mRNA levels in 209 human cell lines representing 16 tumor types were low compared with ESA-responsive positive controls. EpoR protein production was evaluated in a subset of 66 cell lines using a novel anti-EpoR antibody. EpoR(+) control cells had an estimated 10 000 to 100 000 EpoR dimers/cell. In contrast, 54 of 61 lines had EpoR protein levels lower than 100 dimers/cell. Cell lines with the highest EpoR protein levels (400-3200 dimers/cell) were studied further, and, although one line, NCI-H661, bound detectable levels of [(125)I]-recombinant human Epo (rHuEpo), none showed evidence of ESA-induced EpoR activation. There was no increased phosphorylation of STAT5, AKT, ERK, or S6RP with rHuEpo. In addition, EpoR knockdown with siRNAs did not affect viability in 2 cell lines previously reported to express functional EpoR (A2780 and SK-OV-3). These results conflict with the hypothesis that EpoR is functionally expressed in tumors.

Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia.

Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.

Lack of expression and function of erythropoietin receptors in the kidney.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) stimulate formation of red blood cells by binding to and activating Epo receptors (EpoR) on erythroid progenitor cells. Beyond successful treatment of anemia, ESAs have been reported to reduce damage following insult to organs, including the kidney, possibly via direct activation of EpoR. However, data on ESA effects outside hematopoietic functions are conflicting. Furthermore, limited use of appropriate EpoR-positive and EpoR-negative controls and lack of specific anti-EpoR antibodies make interpretation of data difficult. Recently positive and negative control cell types were validated and a sensitive and specific anti-EpoR antibody (A82) that detects low levels of EpoR protein was described. METHODS: A82 was used to measure EpoR protein levels in tissues, human renal cells and human cell lines by western blot analysis. Surface EpoR was examined on renal cells by measuring binding of [125I]-rHuEpo or antibodies. Renal cells and cell lines were treated with rHuEpo to see if phosphorylation of signaling proteins or proliferation/survival could be induced. Small inhibitory RNA (siRNA) were used to determine if EpoR knockdown affected cell viability. RESULTS: Total EpoR protein was low to undetectable in tissues and renal cells with no detectable EpoR on cell surfaces. EpoR knockdown had no effect on viability of renal cell lines. RHuEpo had no detectable effect on intracellular signaling on renal cell lines with no growth-promoting or survival effect on primary human renal cells. CONCLUSIONS: These results suggest that functional EpoR protein is absent on renal cells and that non-EpoR mechanisms should be explored to explain non-hematopoietic effects of ESAs.