Iron biology.

Iron is a fundamental element for biological life, starting from bacteria till humans. Iron is essential for cell function and survival, energy production and metabolism, whereas increased levels cause oxidative stress. It is also a constituent of haemoglobin and thus it is necessary for oxygen transportation through the body. Given these multiple functions, the regulation of iron metabolism is complex and tight coupled with oxygen homeostasis at tissue and cellular levels, thanks to the interaction with the hypoxia inducible factor (HIF) system. In patients with chronic kidney disease (CKD), iron deficiency significantly contributes to anaemia development. This frequently overlaps with chronic inflammation, causing iron- restricted erythropoiesis. To add further complexity, metabolic hyperferritinemia may, on one side, increase the risk for CKD and, on the other, overlaps with functional iron deficiency. Excessive intracellular iron in certain cell types during CKD can also mediate cellular death (called ferroptosis), and contribute to the pathogenesis of kidney damage, atherosclerosis and vascular calcifications. This review is aimed at broadening the perspective of iron metabolism in the setting of CKD not just as a contributor to anaemia in CKD patients, but also as an important player with an impact on cell metabolism, renal fibrosis, and the cardiovascular system.

The anaemia treatment journey of CKD patients: from epoetins to hypoxia-inducible factor-prolyl hydroxylase inhibitors.

The discovery and development of erythropoiesis-stimulating agents was a journey lasting more than a century, leading to the cloning and approval of recombinant human erythropoietin (rHuEpo). This was an impressive clinical advance, providing the possibility of correcting the symptoms associated with anaemia in chronic kidney disease. Associated iron use was needed to produce new haemoglobin-containing blood red cells. Partial anaemia correction became the standard of care since trials aiming for near-normal haemoglobin levels showed a higher risk of adverse cardiovascular events. Hoping to reduce the cardiovascular risks, a new category of drugs was developed and tested. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are small molecules than can be formulated into orally active pills. They simulate reduced tissue oxygen pressure, thus stimulating the production of endogenous erythropoietin (Epo) by the kidneys and liver. Clinical trials with these compounds demonstrated that HIF-PHIs are at least as effective as rHuEpo in treating or correcting anaemia in non-dialysis and dialysis patients. Trials with HIF-PHIs did not demonstrate superiority in safety outcomes and in some trials, outcomes were worse. There was also a focus on oral delivery, a possible beneficial iron-sparing effect and the ability to overcome Epo resistance in inflamed patients. A negative effect is possible iron depletion, which may explain adverse outcomes.

Unified and pluralistic ideals for data sharing and reuse in biodiversity.

How should billions of species observations worldwide be shared and made reusable? Many biodiversity scientists assume the ideal solution is to standardize all datasets according to a single, universal classification and aggregate them into a centralized, global repository. This ideal has known practical and theoretical limitations, however, which justifies investigating alternatives. To support better community deliberation and normative evaluation, we develop a novel conceptual framework showing how different organizational models, regulative ideals and heuristic strategies are combined to form shared infrastructures supporting data reuse. The framework is anchored in a general definition of data pooling as an activity of making a taxonomically standardized body of information available for community reuse via digital infrastructure. We describe and illustrate unified and pluralistic ideals for biodiversity data pooling and show how communities may advance toward these ideals using different heuristic strategies. We present evidence for the strengths and limitations of the unification and pluralistic ideals based on systemic relationships of power, responsibility and benefit they establish among stakeholders, and we conclude the pluralistic ideal is better suited for biodiversity data.

Recognize and Alleviate a Resource Management Conundrum Facing Science Diaspora Networks.

Increasingly, science diaspora networks are managed by formal organizations such as embassies or non-profit organizations. Researchers have studied these networks to understand how they influence international collaborations and science diplomacy, and to determine which network activities foster those outcomes and which do not. In this perspective, we suggest that many of these network organizations confront an underappreciated conundrum for managing resources: organizations with few resources must learn how to obtain more resources despite lacking means to do so. To substantiate our suggestion, we do the following. We review exploratory results from a study of network organizations that indicate that these organizations generally lack resources, learn too little from each other, and struggle to overcome the resource conundrum. We also show that this conundrum is expected from organizational theory based on bounded rationality. To help organizations confront the issue, we do the following. First we provide a new database of operating science diaspora networks. We encourage managers of network organizations to use it as a resource to identify peers with whom to regularly exchange knowledge about securing resources. We also suggest that other scientific organizations should infuse network organizations with fresh resources. Ultimately, we urge all relevant stakeholders to recognize that the conundrum results not from the shortcomings of individual managers, but rather is a legitimate organizational phenomena that must be addressed by organizational design.

Bats, objectivity, and viral spillover risk.

What should the best practices be for modeling zoonotic disease risks, e.g. to anticipate the next pandemic, when background assumptions are unsettled or evolving rapidly? This challenge runs deeper than one might expect, all the way into how we model the robustness of contemporary phylogenetic inference and taxonomic classifications. Different and legitimate taxonomic assumptions can destabilize the putative objectivity of zoonotic risk assessments, thus potentially supporting inconsistent and overconfident policy decisions.

Using participatory action research to examine barriers and facilitators to physical activity among rural adolescents with cerebral palsy.

Purpose: The purpose of this study was to use a qualitative, community-based participatory action research method - Photovoice - to identify perceived facilitators and barriers to physical activity among adolescents with cerebral palsy (CP) in a rural community.Materials and methods: Fifteen participants including adolescents with CP (n = 7) and parents (n = 8) were included in this study. The researchers followed the nine-step methodology recommended for Photovoice. During the training session, participants completed versions of the Barriers to Physical Activity Questionnaire for People with Mobility Impairments. This questionnaire was used to generate descriptive information about participant barriers and facilitators. Participants were given 14 days to take photographs after which researchers used in-depth and focus group interviews structured by the SHOWeD method. Content analysis of transcripts was used to identify common themes.Results and conclusions: Photographs and accompanying text were presented to local stakeholders and an action plan to increase physical activity for adolescents with CP was created. Perceived barriers included lack of inclusiveness, family isolation, and limited accessibility of equipment and resources. Facilitators included support services for families and adaptive sport leagues. Photovoice serves as a powerful tool to initiate change to promote physical activity among rural adolescents with CP.Implications for rehabilitationAdolescents with cerebral palsy living in rural areas face unique barriers to physical activity.Accessibility of equipment and the structural environment can serve as barriers to participation.Lack of accessibility can lead to feelings of isolation.Families need support services outside of rehabilitation settings to support physical activity for their children and overcome potential barriers.

Impact of Inadequate Methods and Data Analysis on Reproducibility.

Failure to reproduce results of articles is recognized, but the causes, and therefore solutions, are not. One possibility is that deficits in quality of the work result in varying or inconclusive results. Erythropoiesis-stimulating agents have been used to treat anemia in patients with cancer, but there are concerns that erythropoiesis-stimulating agents might stimulate Epo receptors on tumor cells (Epo receptor-cancer hypothesis). Articles have been published on the topic, but the data and conclusions conflict, making them suitable for examination of a relationship between quality and reproducibility. Comprehensive literature searches were performed, and 280 relevant articles were identified. Numerous conflicts between and within these articles were apparent. The incidence of faults in quality parameters was high, including absence of adequate controls (90% of articles), inadequate validation of reagents and methods (87% of articles), and inadequate or improper statistical methods (84% of articles) with questionable interpretation of the data (81% of articles). This resulted in false-positive/negative data that varied with the reagents and methods used. The low quality of evidence may explain the poor reproducibility of Epo receptor-cancer articles.

Erythropoiesis stimulating agents and reno-protection: a meta-analysis.

BACKGROUND: Erythropoiesis stimulating agents (ESAs) were proposed to enhance survival of renal tissues through direct effects via activation of EPO receptors on renal cells resulting in reduced cell apoptosis, or indirect effects via increased oxygen delivery due to increased numbers of Hb containing red blood cells. Thus through several mechanisms there may be benefit of ESA administration on kidney disease progression and kidney function in renal patients. However conflicting ESA reno-protection outcomes have been reported in both pre-clinical animal studies and human clinical trials. To better understand the potential beneficial effects of ESAs on renal-patients, meta-analyses of clinical trials is needed. METHODS: Literature searches and manual searches of references lists from published studies were performed. Controlled trials that included ESA treatment on renal patients with relevant renal endpoints were selected. RESULTS: Thirty two ESA controlled trials in 3 categories of intervention were identified. These included 7 trials with patients who had a high likelihood of AKI, 7 trials with kidney transplant patients and 18 anemia correction trials with chronic kidney disease (predialysis) patients. There was a trend toward improvement in renal outcomes in the ESA treated arm of AKI and transplant trials, but none reached statistical significance. In 12 of the anemia correction trials, meta-analyses showed no difference in renal outcomes with the anemia correction but both arms received some ESA treatment making it difficult to assess effects of ESA treatment alone. However, in 6 trials the low Hb arm received no ESAs and meta-analysis also showed no difference in renal outcomes, consistent with no benefit of ESA/ Hb increase. CONCLUSIONS: Most ESA trials were small with modest event rates. While trends tended to favor the ESA treatment arm, these meta-analyses showed no reduction of incidence of AKI, no reduction in DGF or improvement in 1-year graft survival after renal transplantation and no significant delay in progression of CKD. These results do not support significant clinical reno-protection by ESAs.

Bad Science: Cause and Consequence.

Scientific progress is dependent on accumulation of quality data with appropriate data analysis. Unfortunately, there are a troubling number of accounts describing an inability to replicate published work. Some explanations are lack of access to proprietary reagents and equipment, or lack of expertise and know how. However, it is clear that there are many publications that are fatally flawed, and it is difficult to ascertain which ones they are, but there are clues. Many articles are improperly controlled, resulting in false-positive or -negative results. Reagents and procedures are used without verifying their specificity. There is also confirmation bias, a tendency to seek and find conclusions that we like, which is exacerbated by faithful acceptance by readers of the publication record without assessment of merit. These and other issues have slowed progress, resulted in waste of scarce funds, and even put patients at risk when clinical decisions are made according to flawed data. Solving these and related problems requires recognition of the problem and better training. We also need to take personal responsibility for not only our own work, but also for the accuracy of information in the scientific domain.

Functional EpoR pathway utilization is not detected in primary tumor cells isolated from human breast, non-small cell lung, colorectal, and ovarian tumor tissues.

Several clinical trials in oncology have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents. One hypothesis proposes that erythropoiesis-stimulating agents directly stimulate tumor proliferation and/or survival through cell-surface receptors. To test this hypothesis and examine if human tumors utilize the erythropoietin receptor pathway, the response of tumor cells to human recombinant erythropoietin was investigated in disaggregated tumor cells obtained from 186 patients with colorectal, breast, lung, ovarian, head and neck, and other tumors. A cocktail of well characterized tumor growth factors (EGF, HGF, and IGF-1) were analyzed in parallel as a positive control to determine whether freshly-isolated tumor cells were able to respond to growth factor activation ex vivo. Exposing tumor cells to the growth factor cocktail resulted in stimulation of survival and proliferation pathways as measured by an increase in phosphorylation of the downstream signaling proteins AKT and ERK. In contrast, no activation by human recombinant erythropoietin was observed in isolated tumor cells. Though tumor samples exhibited a broad range of cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface erythropoietin receptor was detected in tumor cells from the 186 tumors examined (by flow cytometry or Western blot). Erythropoiesis-stimulating agents did not act directly upon isolated tumor cells to stimulate pathways known to promote proliferation or survival of human tumor cells isolated from primary and metastatic tumor tissues.

Progress in detecting cell-surface protein receptors: the erythropoietin receptor example.

Testing for the presence of specific cell-surface receptors (such as EGFR or HER2) on tumor cells is an integral part of cancer care in terms of treatment decisions and prognosis. Understanding the strengths and limitations of these tests is important because inaccurate results may occur if procedures designed to prevent false-negative or false-positive outcomes are not employed. This review discusses tests commonly used to identify and characterize cell-surface receptors, such as the erythropoietin receptor (EpoR). First, a summary is provided on the biology of the Epo/EpoR system, describing how EpoR is expressed on erythrocytic progenitors and precursors in the bone marrow where it mediates red blood cell production in response to Epo. Second, studies are described that investigated whether erythropoiesis-stimulating agents could stimulate tumor progression in cancer patients and whether EpoR is expressed and functional on tumor cells or on endothelial cells. The methods used in these studies included immunohistochemistry, Northern blotting, Western blotting, and binding assays. This review summarizes the strengths and limitations of these methods. Critically analyzing data from tests for cell-surface receptors such as EpoR requires understanding the techniques utilized and demonstrating that results are consistent with current knowledge about receptor biology.

Dose-related differences in the pharmacodynamic and toxicologic response to a novel hyperglycosylated analog of recombinant human erythropoietin in Sprague-Dawley rats with similarly high hematocrit.

We recently reported results that erythropoiesis-stimulating agent (ESA)-related thrombotic toxicities in preclinical species were not solely dependent on a high hematocrit (HCT) but also associated with increased ESA dose level, dose frequency, and dosing duration. In this article, we conclude that sequelae of an increased magnitude of ESA-stimulated erythropoiesis potentially contributed to thrombosis in the highest ESA dose groups. The results were obtained from two investigative studies we conducted in Sprague-Dawley rats administered a low (no thrombotic toxicities) or high (with thrombotic toxicities) dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114), 3 times weekly for up to 9 days or for 1 month. Despite similarly increased HCT at both dose levels, animals in the high-dose group had an increased magnitude of erythropoiesis measured by spleen weights, splenic erythropoiesis, and circulating reticulocytes. Resulting prothrombotic risk factors identified predominantly or uniquely in the high-dose group were higher numbers of immature reticulocytes and nucleated red blood cells in circulation, severe functional iron deficiency, and increased intravascular destruction of iron-deficient reticulocyte/red blood cells. No thrombotic events were detected in rats dosed up to 9 days suggesting a sustained high HCT is a requisite cofactor for development of ESA-related thrombotic toxicities.

Cytokines associated with increased erythropoiesis in Sprague-Dawley rats administered a novel hyperglycosylated analog of recombinant human erythropoietin.

We previously reported an increased incidence of thrombotic toxicities in Sprague-Dawley rats administered the highest dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114) for 1 month as not solely dependent on high hematocrit (HCT). Thereafter, we identified increased erythropoiesis as a prothrombotic risk factor increased in the AMG 114 high-dose group with thrombotic toxicities, compared to a low-dose group with no toxicities but similar HCT. Here, we identified pleiotropic cytokines as prothrombotic factors associated with AMG 114 dose level. Before a high HCT was achieved, rats in the AMG 114 high, but not the low-dose group, had imbalanced hemostasis (increased von Willebrand factor and prothrombin time, decreased antithrombin III) coexistent with cytokines implicated in thrombosis: monocyte chemotactic protein 1 (MCP-1), MCP-3, tissue inhibitor of metalloproteinases 1, macrophage inhibitory protein-2, oncostatin M, T-cell-specific protein, stem cell factor, vascular endothelial growth factor, and interleukin-11. While no unique pathway to erythropoiesis stimulating agent-related thrombosis was identified, cytokines associated with increased erythropoiesis contributed to a prothrombotic intravascular environment in the AMG 114 high-dose group, but not in lower dose groups with a similar high HCT.

Epo receptors are not detectable in primary human tumor tissue samples.

Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment, low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82), little/no EpoR protein was detected and it was not functional. In contrast, EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20), and functional responses to rHuEpo were reported with MCF-7 cells. In another study, a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However, the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82, no EpoR protein was detectable in normal human and matching cancer tissues from breast, lung, colon, ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells, but no rHuEpo-induced phosphorylation of AKT, STAT3, pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein.

The effect of erythropoietin on normal and neoplastic cells.

Erythropoietin (Epo) is an essential hormone that binds and activates the Epo receptor (EpoR) resident on the surface of erythroid progenitor cells, thereby promoting erythropoiesis. Recombinant human erythropoietin has been used successfully for over 20 years to treat anemia in millions of patients. In addition to erythropoiesis, Epo has also been reported to have other effects, such as tissue protection and promotion of tumor cell growth or survival. This became of significant concern in 2003, when some clinical trials in cancer patients reported increased tumor progression and worse survival outcomes in patients treated with erythropoiesis-stimulating agents (ESAs). One of the potential mechanisms proffered to explain the observed safety issues was that functional EpoR was expressed in tumors and/or endothelial cells, and that ESAs directly stimulated tumor growth and/or antagonized tumor ablative therapies. Since then, numerous groups have performed further research evaluating this potential mechanism with conflicting data and conclusions. Here, we review the biology of endogenous Epo and EpoR expression and function in erythropoiesis, and evaluate the evidence pertaining to the expression of EpoR on normal nonhematopoietic and tumor cells.

Abuse of medicines for performance enhancement in sport: why is this a problem for the pharmaceutical industry?

The misuse of medicines for performance enhancement in sport (doping) is not approved by regulatory agencies, and is illegal in many countries. In addition to the 'traditional' doping agents such as steroids, ß-blockers and blood transfusions, the list of agents and techniques used in doping is increasing and now includes newer medicines such as erythropoiesis-stimulating agents and growth hormones. Innovative new medicines are of particular interest as would-be dopers may believe them to be undetectable by current methods. Close collaboration between the biopharmaceutical industry and anti-doping agencies such as the World Anti-Doping Agency is critical to a successful anti-doping strategy. Industry is ideally placed to identify the doping potential of new medicines at early stages and to support early development of detection assays. A strong, united front between the biopharmaceutical industry and anti-doping agencies is essential to counter the misuse of medicines for performance enhancement, as well as to promote fair play and clean sport.

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.

Using the dimensions of health to assess motivation among running moms.

The Centers for Disease Control and Prevention (2008) reported that 60% of American women did not engage in the recommended daily amount of physical activity and 25% are not physically active at all. The concept of holistic health or wellness refers to an active process in which an individual's life is influenced by dimensions that include physical, emotional, social, mental, and spiritual factors.

Erythropoiesis-stimulating agents.

Erythropoiesis is the process whereby erythroid progenitor cells differentiate and divide, resulting in increased numbers of red blood cells (RBCs). RBCs contain hemoglobin, the main oxygen carrying component in blood. The large number of RBCs found in blood is required to support the prodigious consumption of oxygen by tissues as they undergo oxygen-dependent processes. Erythropoietin is a hormone that when it binds and activates Epo receptors resident on the surface of cells results in stimulation of erythropoiesis. Successful cloning of the EPO gene allowed for the first time production of recombinant human erythropoietin and other erythropoiesis stimulating agents (ESAs), which are used to treat anemia in patients. In this chapter, the control of Epo levels and erythropoiesis, the various forms of ESAs used commercially, and their physical and biological properties are discussed.