Many disorders of iron homeostasis (e.g., iron overload) are associated with the dynamic kinetic profiles of multiple non-transferrin bound iron (NTBI) species, chronic exposure to which is associated with deleterious end-organ effects. Here we discuss the chemical nature of NTBI species, challenges with measuring NTBI in plasma, and the clinical relevance of NTBI exposure based on source (iron overload disorder vs. intravenous iron-carbohydrate complex administration). NTBI is not a single entity but consists of multiple, often poorly characterized species, some of which are kinetically non-exchangeable while others are relatively exchangeable. Prolonged presence of plasma NTBI is associated with excessive tissue iron accumulation in susceptible tissues, with consequences, such as endocrinopathy and heart failure. In contrast, intravenous iron-carbohydrate nanomedicines administration leads only to transient NTBI appearance and lacks evidence for association with adverse clinical outcomes. Assays to measure plasma NTBI are typically technically complex and remain chiefly a research tool. There have been two general approaches to estimating NTBI: capture assays and redox-activity assays. Early assays could not avoid capturing some iron from transferrin, thus overestimating NTBI. By contrast, some later assays may have promoted the donation of NTBI species to transferrin during the assay procedure, potentially underestimating NTBI levels. The levels of transferrin saturation at which NTBI species have been detectable have varied between different methodologies and between patient populations studied.

Hemochromatosis (HC) is a genetically heterogeneous disorder in which uncontrolled intestinal iron absorption may lead to progressive iron overload (IO) responsible for disabling and life-threatening complications such as arthritis, diabetes, heart failure, hepatic cirrhosis, and hepatocellular carcinoma. The recent advances in the knowledge of pathophysiology and molecular basis of iron metabolism have highlighted that HC is caused by mutations in at least 5 genes, resulting in insufficient hepcidin production or, rarely, resistance to hepcidin action. This has led to an HC classification based on different molecular subtypes, mainly reflecting successive gene discovery. This scheme was difficult to adopt in clinical practice and therefore needs revision. Here we present recommendations for unambiguous HC classification developed by a working group of the International Society for the Study of Iron in Biology and Medicine (BIOIRON Society), including both clinicians and basic scientists during a meeting in Heidelberg, Germany. We propose to deemphasize the use of the molecular subtype criteria in favor of a classification addressing both clinical issues and molecular complexity. Ferroportin disease (former type 4a) has been excluded because of its distinct phenotype. The novel classification aims to be of practical help whenever a detailed molecular characterization of HC is not readily available.

Elevated levels of mitochondrial iron and reactive oxygen species (ROS) accompany the progression of diabetes, negatively impacting insulin production and secretion from pancreatic cells. In search for a tool to reduce mitochondrial iron and ROS levels, we arrived at a molecule that destabilizes the [2Fe-2S] clusters of NEET proteins (M1). Treatment of db/db diabetic mice with M1 improved hyperglycemia, without the weight gain observed with alternative treatments such as rosiglitazone. The molecular interactions of M1 with the NEET proteins mNT and NAF-1 were determined by X-crystallography. The possibility of controlling diabetes by molecules that destabilize the [2Fe-2S] clusters of NEET proteins, thereby reducing iron-mediated oxidative stress, opens a new route for managing metabolic aberration such as in diabetes.

Considered a key aging gene, CISD2 , encoding CDGSH iron–sulfur domain‐containing protein 2, plays a central role in regulating calcium homeostasis, preventing mitochondrial dysfunction, and the activation of autophagy and apoptosis in different cells. Here, we show that cardiomyocytes from CISD2 ‐null mice accumulate high levels of iron and contain high levels of transferrin receptor and ferritin. Using proteomics and transmission electron microscopy, we further show that the lack of CISD2 induces several features of the aging process in young mice, but other features are not induced. Taken together, our findings suggest that CISD2 protects cardiomyocytes from overaccumulation of iron, which is common in aging hearts and can contribute to the pathogenesis of heart failure.

Decreased insulin secretion, associated with pancreatic β-cell failure, plays a critical role in many human diseases including diabetes, obesity, and cancer. While numerous studies linked β-cell failure with enhanced levels of reactive oxygen species (ROS), the development of diabetes associated with hereditary conditions that result in iron overload, e.g., hemochromatosis, Friedreich's ataxia, and Wolfram syndrome type 2 (WFS-T2; a mutation in CISD2, encoding the [2Fe-2S] protein NAF-1), underscores an additional link between iron metabolism and β-cell failure. Here, using NAF-1-repressed INS-1E pancreatic cells, we observed that NAF-1 repression inhibited insulin secretion, as well as impaired mitochondrial and ER structure and function. Importantly, we found that a combined treatment with the cell permeant iron chelator deferiprone and the glutathione precursor N-acetyl cysteine promoted the structural repair of mitochondria and ER, decreased mitochondrial labile iron and ROS levels, and restored glucose-stimulated insulin secretion. Additionally, treatment with the ferroptosis inhibitor ferrostatin-1 decreased cellular ROS formation and improved cellular growth of NAF-1 repressed pancreatic cells. Our findings reveal that suppressed expression of NAF-1 is associated with the development of ferroptosis-like features in pancreatic cells, and that reducing the levels of mitochondrial iron and ROS levels could be used as a therapeutic avenue for WFS-T2 patients.

Vaccines are the most effective measure to prevent deaths and illness from infectious diseases. Nevertheless, the efficacy of several paediatric vaccines is lower in low-income and middle-income countries (LMICs), where mortality from vaccine-preventable infections remains high. Vaccine efficacy can also be decreased in adults in the context of some common comorbidities. Identifying and correcting the specific causes of impaired vaccine efficacy is of substantial value to global health. Iron deficiency is the most common micronutrient deficiency worldwide, affecting more than 2 billion people, and its prevalence in LMICs could increase as food security is threatened by the COVID-19 pandemic. In this Viewpoint, we highlight evidence showing that iron deficiency limits adaptive immunity and responses to vaccines, representing an under-appreciated additional disadvantage to iron deficient populations. We propose a framework for urgent detailed studies of iron-vaccine interactions to investigate and clarify the issue. This framework includes retrospective analysis of newly available datasets derived from trials of COVID-19 and other vaccines, and prospective testing of whether nutritional iron interventions, commonly used worldwide to combat anaemia, improve vaccine performance.

American Journal of HematologyVolume 97, Issue 1 p. 7-9 COMMENTARYFree Access Plasma nontransferrin bound iron–nontransferrin bound iron revisited: Implications for systemic iron overload and in iv iron supplementation Ioav Z. Cabantchik, Corresponding Author Ioav Z. Cabantchik [email protected] orcid.org/0000-0001-8638-3142 Institute of Life Sciences, Faculty of Natural Sciences, Hebrew University of Jerusalem, Jerusalem, Israel Correspondence Ioav Z. Cabantchik. Alexander Silberman Institute of Life Sciences, Department of Biological Chemistry, Safra Campus at Givar Ram, Jerusalem 91904, Israel. Email: [email protected]Search for more papers by this authorChaim Hershko, Chaim Hershko Institute of Medicine, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, IsraelSearch for more papers by this author Ioav Z. Cabantchik, Corresponding Author Ioav Z. Cabantchik [email protected] orcid.org/0000-0001-8638-3142 Institute of Life Sciences, Faculty of Natural Sciences, Hebrew University of Jerusalem, Jerusalem, Israel Correspondence Ioav Z. Cabantchik. Alexander Silberman Institute of Life Sciences, Department of Biological Chemistry, Safra Campus at Givar Ram, Jerusalem 91904, Israel. Email: [email protected]Search for more papers by this authorChaim Hershko, Chaim Hershko Institute of Medicine, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, IsraelSearch for more papers by this author First published: 12 October 2021 https://doi.org/10.1002/ajh.26374AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Nontransferrin bound iron (NTBI) has been classically used to denote a potentially toxic component of plasma detected in systemic iron overload and implicated in end-organ iron accumulation and biological damage. A chelatable labile component of plasma NTBI has also served as target for novel therapeutics. However, as the term NTBI per se does not exclude nontoxic forms of iron that can be detected in plasma, its liberal use without additional pharmacological attributes might be misleading, especially in the clinical context, as for infusible iron supplements. In keeping with the historical and commonly used NTBI term, we propose here to use "labile/nonlabile" plasma NTBI to indicate the toxic potential of the iron species in question. Iron overload (IO) is a pathological condition caused by the accumulation of iron in excess of physiological needs, ultimately resulting in oxidative damage to vital functions. The early diagnosis of the most prevalent IO pathologies has classically relied on the two systemic iron plasma markers, ferritin, and transferrin saturation (TSAT). The end-organ accumulation of iron and ensuing damage were attributed to the opportunistic ingress of nontransferrin bound iron (NTBI) species from plasma to cells, via undefined membrane pathways. The early definition of NTBI in IO plasma (TSAT ≥ 100%) was of an iron species of relatively low molecular weight iron that is not associated with the physiological iron carrier transferrin, insofar as it was captured by a chelator and filterable via dialysis.1, 2 The putative NTBI species was subsequently referred to as plasma catalytic iron, based on its propensity to generate reactive oxygen species (ROS), a property commonly implicated in peroxidative damage.3 1 NTBI-ORIGIN AND TERMINOLOGY Although NTBI per se implies generically all forms of iron not-bound to transferrin, its usage in biology and medicine (especially in hematology), has been confined to either chelatable or catalytic iron in internal biological fluids (reviewed in Brissot et al., 2012. doi: 0.1016/j.bbagen.2011.07.014 and Cabantchik, 2014. doi: 10.3389/fphar.2014.00045).*4, 5 As NTBI was detected in plasma of patients with IO (TSAT>70%), it was viewed as a marker of systemic IO but also inferred as source of impending tissue IO, namely as a potential source of unregulated tissue iron ingress and ensuing accumulation to toxic levels (see also review by Porter & Garbowski, 2014. doi: 10.1016/j.hoc.2014.04.003).6 Although the suggested origin of tissue IO and peroxidative damage was identified with the fraction of plasma iron not associated with transferrin, the role of plasma NTBI in IO pathology was essentially deduced from the protective effect afforded by iron chelators with limited permeation to cells. The ability of chelators to complex plasma NTBI, was subsequently used in the treatment of systemic IO to assess chelation efficacy by different chelation regimens. The generation of NTBI in human plasma reflects the failure of transferrin to capture all iron outpouring into circulation via the ferroportin export system. Transferrin's failure results primarily from a "local insufficiency" of apotransferrin (measurable as UIBC-unsaturated iron-binding capacity) due primarily to the lack of transferrin as in hereditary aceruloplasminemia, liver failure, or insufficient apotransferrin or when iron is under-utilized by the hematopoietic system (see also review by Coates, 2014. doi: 10.1016/j.freeradbiomed.2014.03.039).7 As NTBI is generally removed from circulation by the liver, its plasma levels reflect the dynamics of NTBI generation vis a vis its removal from plasma. Thus, NTBI builds up whenever production exceeds removal from circulation, as found in various types of IO of transfusional origin but also in nontransfusional overload that results from hyperabsorption of iron as in hereditary hemochromatosis or thalassemia intermedia, various forms of ineffective or suppressed erythropoiesis and especially in atransferrinemia). 2 NTBI-A PARAMETER OF IO AND IMPENDING TOXICITY Plasma NTBI was originally detected in patients with systemic IO from different origins and the peroxidative organ damage caused was generally attributed to the uncontrolled infiltration of NTBI into cells. However, the threshold levels of plasma NTBI potential toxicity still remain undefined in the different clinical settings. First and foremost, it has been difficult to identify the chemical composition of the plasma NTBI toxic species that infiltrate different cells opportunistically, their concentrations, and the periods required for them to accumulate in different cell types to the respective toxic levels (as those depend also on the individual routes of iron infiltration and cell defense mechanisms). Second, various forms of NTBI can be detected in plasma not only in overt IO8 but also in normal individuals,9 clearly indicating that the putative NTBI per se need not be pathological or necessarily noxious, paraphrasing Paracelsus.† Notably, the existence of detectable NTBI in the plasma of normal individuals might explain why some NTBI assays, particularly those that assess labile plasma iron (LPI), detect a basal level of labile NTBI in normal plasma.9, 10 Although the use of NTBI was initially confined to a potentially toxic entity in plasma of IO individuals, the term NTBI per se did not exclude plasma ferritin-iron detected in IO plasma, nor iatrogenic NTBI-chelates resulting from the treatment of IO with iron chelators or NTBI-complexes given by intravenous infusions (primarily ferric oxyhydroxide copolymerized with carbohydrate moieties).‡ All those compounds detected in human plasma fall in the category of plasma NTBI, irrespective of their chemical reactivity or toxic potential.§ However, over the years, the NTBI term has been used more liberally, far from what was originally intended, including any iron-complex applied experimentally for loading cells with iron. In many cases, those compounds were implied to simulate/mimic the genuine pathological NTBIs, whose chemical structures are yet to be fully resolved, especially those germane to pathological tissue IO. We are of the idea that in the biomedical setting, the term NTBI¶ should be used in a way that will reflect its pathological nature and toxic potential. For that, a clear distinction between potentially toxic and nontoxic NTBI species would seem timely and appropriate, for scientific, diagnostic as for therapeutic reasons. Whether any chemical agent is or is not toxic was addressed by Paracelsus for more than 500 years in reference to poisons. The criterion is empirical and mostly based on the dose, but also on the period exposure to the toxic agent in the native (or closely similar) biological milieu, be it cell or whole organism. Iron toxicity stems primarily from its chemical lability when complexed to particular ligands that modulate its redox potential, particularly its reactivity in an oxygenated environment. 3 A PROPOSAL We propose to continue using the generic NTBI, mainly for historical reasons, but add to NTBI a qualifier to specify its labile or nonlabile character, as that qualifier refers directly to either its toxic or nontoxic potential, respectively. For example: "labile-NTBI" refers to iron that is transferable/chelatable and redox-active, rendering "plasma labile-NTBI" and "labile plasma iron" as equivalent terms applicable to the clinical setting of systemic IO (e.g. NTBI species with potential toxicity, as found in the plasma of IO patients with hemochromatosis, secondary IO resulting from repeated blood transfusions as in thalassemia syndromes, or in patients following myeloablative chemotherapy and others). Similarly, plasma NTBI derived from infusible iron supplements containing ferric oxyhydroxide-carbohydrate, or from fully coordinated iron-chelates following chelation treatment, should be referred to as nonlabile NTBI or rather demonstrably free of labile NTBI, irrespective of their chemical structure. 4 WHAT IS NTBI, HOW TO PRACTICALLY ASSESS IT IN PLASMA, AND IS IT OF ANY CLINICAL USE? Hitherto the chemical identity of plasma labile-NTBI species remains to be determined in different IO states that differ in their plasma chemical composition, the degree, and speed of IO and the redox activity of the medium. Although NTBI speciation studies indicated a range of iron citrate species as likely candidates,8, 11 the actual forms of labile-NTBI relevant to pathological loading of tissue compartments across disease states need to be elucidated, particularly for characterizing the membrane routes of iron loading into different tissues (reviewed by Knutson, 2019. doi: 10.1016/j.freeradbiomed.2018.10.413).12 The detection of plasma NTBI has relied on analytical assays that monitor iron captured (chelated) by a metal sensitive probe or by measuring iron's redox catalytic activity that is susceptible to chelation. "Total" NTBI detected in plasma provides a measure of IO, while potentially toxic or labile fractions have been detected by LPI assays. Importantly, the notion that tissue IO could be reduced by controlling labile-NTBI levels in plasma, has led to the development of clinical chelator regimens aimed at minimizing daily tissue exposure to plasma labile-NTBI components.11 NTBI remains an important concept in clinical management of systemic IO, because much of the morbidity and mortality in systemic IO have been attributed to excess tissue iron accumulation from overexposure to NTBI. However, its limited use in clinical research stems from outstanding need for validating assays for quantitating plasma NTBI species in different IO pathologies and defining their respective etio-pathological roles (including threshold toxic levels). Those shortcomings are also reflected in the NTBI values reported by different analytical assays performed on the same plasma of patients with different sources of IO13 and different values of labile iron reported for iv-iron formulations using different assays, including values in plasma following iv administration.14 The differences stem not only from variable sensitivity and/or specificity and/or robustness of the NTBI assays, but also from the choice of reference standards used for measuring chemically heterogeneous species taken from a complex-heterogeneous medium, that is, plasma or serum. Hitherto, the major clinical contribution of labile-NTBI assays has been in the design of treatment regimens that aim to minimize tissue exposure to plasma NTBI components (e.g. LPI) and hence provide extended protection from impending IO. Initial attempts have been done to determine the threshold levels of labile NTBI/LPI or TSAT that could serve as indicators for initiating chelation in naïve hypertranfused children with thalassemia major.15 For diagnostic purposes, labile NTBI/LPI > 0.2 μM levels were consistently found in plasma with TSAT levels >70% and maintaining levels below this throughout the day in IO patients can serve as one of the goals in the treatment of IO or vice versa, to start treatment (particularly chelation) when threshold levels of NTBI/LPI or TSAT are surpassed. 5 CONCLUSIONS AND PERSPECTIVES While plasma NTBI remains an important pathological marker of IO and potential organ toxicity, the lack of clinical validation of "toxic"-plasma levels NTBI/LPI has thus far limited its use in clinical practice. A simple, robust validated assay is still to be established for carrying out large-scale clinical studies in order to determine the degree of IO (as it is reflected in labile NTBI/LPI and TSAT levels), primarily for initiating and/or optimizing therapeutic interventions. As in the case of serum cholesterol, the journey to achieve that goal might be long and treacherous. However, "The danger for most of us lies not in setting our aim too high and falling short, but in setting our aim too low, and achieving our mark."-Michelangelo Buonarroti. As to the biomedical use of the term NTBI for any nonphysiological compound applied to or detected in a transferrin-containing medium, we urge to specify its labile/nonlabile chemical character to denote its toxic potential. Endnotes * NTBI is often referred as "free iron" to denote iron not bound to physiological transferrin or to ferritin or heme. † From the father of Toxicology: "What is there that is not poison? "wrote Paracelsus 500 years ago. "All things are poison and nothing is without poison. Solely the dose determines that a thing is not a poison" he clarified. However, it is more than just the dose, is also a matter of time of exposure to a given dose and of environmental factors. ‡ The name Food and Drug Administration (FDA) suggested for iron in commercial iron-carbohydrate formulations used for iv-supplementation. § Technically, NTBI assays per se do not necessarily distinguish between NTBI with different toxic potential. ¶ The term NTBI is apophatic as it "describes something by stating which characteristics it does not have" (Merriam-Webster Dictionary). REFERENCES 1Hershko C, Graham G, Bates GW, Rachmilewitz EA. Non-specific serum iron in thalassaemia: an abnormal serum iron fraction of potential toxicity. Br J Haematol. 1978; 40: 255- 263. doi:10.1111/j.1365-2141.1978.tb03662.x 2Gosriwatana I, Loreal O, Lu S, Brissot P, Porter J, Hider RC. Quantification of non-transferrin-bound iron in the presence of unsaturated transferrin. Anal Biochem. 1999; 273: 212- 220. doi:10.1006/abio.1999.4216 3Gutteridge JM, Rowley DA, Griffiths E, Halliwell B. Low-molecular-weight iron complexes and oxygen radical reactions in idiopathic haemochromatosis. Clin Sci (Lond). 1985; 68: 463- 467. doi:10.1042/cs0680463 4Brissot P, Ropert M, Le Lan C, Loréal O. Non-transferrin bound iron: a key role in iron overload and iron toxicity. Biochim Biophys Acta. 2012; 1820: 403- 410. doi:10.1016/j.bbagen.2011.07.014 5Cabantchik Z. Labile iron in cells and body fluids: physiology, pathology, and pharmacology. Front Pharmacol. 2014; 5: 45. doi:10.3389/fphar.2014.00045 6Porter JB, Garbowski M. The pathophysiology of transfusional iron overload. Hematol Oncol Clin North Am. 2014; 28: 683- 701. doi:10.1016/j.hoc.2014.04.003 7Cotes TD. Physiology and Pathophysiology of Iron in Hemoglobin-Associated Diseases. Free Radic Biol Med. 2014; 72: 23- 40. doi:10.1016/j.freeradbiomed.2014.03.039 8Evans RW, Rafique R, Zarea A, et al. Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera. J Biol Inorg Chem. 2008; 13: 57- 74. doi:10.1007/s00775-007-0297-8 9Dziuba N, Hardy J, Lindahl PA. Low-molecular-mass iron in healthy blood plasma is not predominately ferric citrate. Metallomics. 2018; 10: 802- 817. doi:10.1039/c8mt00055g 10Breuer W, Ghoti H, Shattat A, et al. Non-transferrin bound iron in thalassemia: differential detection of redox active forms in children and older patients. Am J Hematol. 2012; 87: 55- 61. doi:10.1002/ajh.22203 11Porter JB, El-Alfy M, Viprakasit V, et al. Utility of labile plasma iron and transferrin saturation in addition to serum ferritin as iron overload markers in different underlying anemias before and after deferasirox treatment. Eur J Haematol. 2016; 96: 19- 26. doi:10.1111/ejh.12540 12Knutson MD. Non-transferrin-bound iron transporters. Free Radic Biol Med. 2019; 133: 101- 111. doi:10.1016/j.freeradbiomed.2018.10.413 13de Swart L, Hendriks JC, van der Vorm LN, et al. Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders. Haematologica. 2016; 101: 38- 45. doi:10.3324/haematol.2015.133983 14Garbowski MW, Bansal S, Porter JB, Mori C, Burckhardt S, Hider RC. Intravenous iron preparations transiently generate non-transferrin-bound iron from two proposed pathways. Haematologica. 2020. doi:10.3324/haematol.2020.250803 15Danjou F, Cabantchik ZI, Origa R, et al. A decisional algorithm to start iron chelation in patients with beta thalassemia. Haematologica. 2014; 99: e38- e40. doi:10.3324/haematol.2013.098202 Volume97, Issue1January 2022Pages 7-9 ReferencesRelatedInformation

Focal iron accumulation associated with brain iron dyshomeostasis is a pathological hallmark of various neurodegenerative diseases (NDD). The application of iron-sensitive sequences in magnetic resonance imaging has provided a useful tool to identify the underlying NDD pathology. In the three major NDD, degeneration occurs in central nervous system (CNS) regions associated with memory (Alzheimer's disease, AD), automaticity (Parkinson's disease, PD) and motor function (amyotrophic lateral sclerosis, ALS), all of which require a high oxygen demand for harnessing neuronal energy. In PD, a progressive degeneration of the substantia nigra pars compacta (SNc) is associated with the appearance of siderotic foci, largely caused by increased labile iron levels resulting from an imbalance between cell iron import, storage and export. At a molecular level, α-synuclein regulates dopamine and iron transport with PD-associated mutations in this protein causing functional disruption to these processes. Equally, in ALS, an early iron accumulation is present in neurons of the cortico-spinal motor pathway before neuropathology and secondary iron accumulation in microglia. High serum ferritin is an indicator of poor prognosis in ALS and the application of iron-sensitive sequences in magnetic resonance imaging has become a useful tool in identifying pathology. The molecular pathways that cascade down from such dyshomeostasis still remain to be fully elucidated but strong inroads have been made in recent years. Far from being a simple cause or consequence, it has recently been discovered that these alterations can trigger susceptibility to an iron-dependent cell-death pathway with unique lipoperoxidation signatures called ferroptosis. In turn, this has now provided insight into some key modulators of this cell-death pathway that could be therapeutic targets for the NDD. Interestingly, iron accumulation and ferroptosis are highly sensitive to iron chelation. However, whilst chelators that strongly scavenge intracellular iron protect against oxidative neuronal damage in mammalian models and are proven to be effective in treating systemic siderosis, these compounds are not clinically suitable due to the high risk of developing iatrogenic iron depletion and ensuing anaemia. Instead, a moderate iron chelation modality that conserves systemic iron offers a novel therapeutic strategy for neuroprotection. As demonstrated with the prototype chelator deferiprone, iron can be scavenged from labile iron complexes in the brain and transferred (conservatively) either to higher affinity acceptors in cells or extracellular transferrin. Promising preclinical and clinical proof of concept trials has led to several current large randomized clinical trials that aim to demonstrate the efficacy and safety of conservative iron chelation for NDD, notably in a long-term treatment regimen.

Despite major advances in Biology and Medicine and the increasing awareness of nutrition to human health and wellness, anemia remains a major public health problem. About half of the millions of anemia cases worldwide are associated with iron deficiency (ID) that manifest initially as physical fatigue and progressively leads to major dysfunctions with serious clinical outcomes. A major source of the problem is inadequate (ineffective or insufficient) food sources, that exacerbates during worldwide crisis, as in the present corona pandemics. Those affected are primarily children, young girls, pregnant women and the elder population. But among those most afflicted are individuals with chronic inflammatory disorders, as found in inflammatory bowel disorders IBD (Crohn’s, ulcerative colitis, etc), in celiac, rheumatoid arthritis, most types of cancer and especially in infections (bacterial, viral or parasitic). How does one clinically cope with chronic ID in normal times and most importantly during periods of crisis, when clinical services relevant to the ID population are limited or inaccessible?

Iron accumulation has been observed in mouse models and in both sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Iron chelation could reduce iron accumulation and the related excess of oxidative stress in the motor pathways. However, classical iron chelation would induce systemic iron depletion. We assess the safety and efficacy of conservative iron chelation (i.e., chelation with low risk of iron depletion) in a murine preclinical model and pilot clinical trial. In Sod1(G86R) mice, deferiprone increased the mean life span compared with placebo. The safety was good, without anemia after 12 months of deferiprone in the 23 ALS patients enrolled in the clinical trial. The decreases in the ALS Functional Rating Scale and the body mass index were significantly smaller for the first 3 months of deferiprone treatment (30 mg/kg/day) than for the first treatment-free period. Iron levels in the cervical spinal cord, medulla oblongata, and motor cortex (according to magnetic resonance imaging), as well as cerebrospinal fluid levels of oxidative stress and neurofilament light chains were lower after deferiprone treatment. Our observation leads to the hypothesis that moderate iron chelation regimen that avoids changes in systemic iron levels may constitute a novel therapeutic modality of neuroprotection for ALS. Antioxid. Redox Signal. 00, 000-000.

Universit e de Lille, CHU de Lille, INSERM UMRS_1171, Service de Neurologie NS-Park/FCRIN Network LICEND COEN Center, Lille, France School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, West Yorkshire, UK, and Oxidation Biology Unit, Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia Universit e de Toulouse, UPS, CHU de Toulouse, INSERM; Centre d’Investigation Clinique CIC1436, Services de Neurologie et de Pharmacologie Clinique, UMR TONIC, NS-Park/FCRIN Network, NeuroToul COEN Center, Toulouse, France University de Lille, CHU de Lille, INSERM UMRS_1171, NS-Park/FCRIN Network LICEND COEN Center, Lille, France Department of Neurology, Christian-Albrechts-University of Kiel, Kiel, Germany and Hertie-Institute of Clinical Brain Research, Department of Neurodegeneration, T€ ubingen, Germany Imperial College London, London, UK Della Pergola Chair, Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel Oxidation Biology Unit, Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia Universit e de Lille, CHU de Lille, INSERM UMRS_1171, Service de Pharmacologie Clinique et Service de Neurologie NS-Park/FCRIN Network LICEND COEN Center, Lille, France

Poor adherence of transfusion-dependent patients to chelation treatment is often the cause of persistent iron overload and ensuing morbidity. However, a tool to assess patient compliance with therapy is lacking in clinical practice. Labile plasma iron (LPI, the redox-active component of non-transferrin bound iron) has been studied as an indicator of systemic iron overload and of chelation efficacy, and may particularly reflect recent iron equilibrium. We considered the use of LPI as a potential indicator for recent chelation treatment in 18 transfusion-dependent pediatric patients. Samples were collected under chelation treatment or after a short interruption of the treatment, and LPI was measured by the FeROS assay (Aferrix, Tel Aviv, Israel). LPI was significantly higher after a short-term interruption of the chelation (median of 0.4 mu M off-therapy [range:0-4] vs 0 mu M on-therapy [range:0-2.8] (p<.001)). Conversely, serum iron, serum ferritin and calculated transferrin saturation were not significantly higher in the "off-therapy" samples compared to "on-therapy". In addition, in multivariate logistic regression analysis LPI was the variable most significantly associated with recent chelation treatment (p=.001). We conclude that LPI could serve as a useful indicator of compliance to chelation therapy.

SIGNIFICANCECancer cells accumulate high levels of iron and reactive oxygen species (ROS) to promote their high metabolic activity and proliferation rate. However, high levels of iron and ROS can also lead to enhanced oxidative stress and the activation of cell death pathways such as apoptosis and ferroptosis. This has led to the proposal that different drugs that target iron and/or ROS metabolism could be used as anticancer drugs. However, due to the complex role iron and ROS play in cells, the majority of these drugs yielded mixed results, highlighting a critical need to identify new players in the regulation of iron and ROS homeostasis in cancer cells. Recent Advances: NEET proteins belong to a newly discovered class of iron-sulfur proteins (2Fe-2S) required for the regulation of iron and ROS homeostasis in cells. Recent studies revealed that the NEET proteins NAF-1 (CISD2) and mitoNEET (CISD1) play a critical role in promoting the proliferation of cancer cells, supporting tumor growth and metastasis. Moreover, the function of NEET proteins in cancer cells was found to be dependent of the degree of lability of their 2Fe-2S clusters.CRITICAL ISSUESNEET proteins could represent a key regulatory link between the maintenance of high iron and ROS in cancer cells, the activation of cell death and survival pathways, and cellular proliferation.FUTURE DIRECTIONSBecause the function of NEET proteins depends on the lability of their clusters, drugs that target the 2Fe2S clusters of NEET proteins could be used as promising anticancer drugs.

Although guidelines are available for hereditary hemochromatosis, a high percentage of the recommendations within them are not shared between the different guidelines. Our main aim is to provide an objective, simple, brief, and practical set of recommendations about therapeutic aspects of HFE hemochromatosis for p.Cys282Tyr (C282Y/C282Y) homozygous genotype, based on the published scientific studies and guidelines, in a form that is reasonably comprehensible to patients and people without medical training. This final version was approved at the Hemochromatosis International meeting on 12th May 2017 in Los Angeles.

Eliezer Rachmilewitz (1935–2017).The hematology community mourns the sudden death of Professor Eliezer Rachmilewitz, who passed away on December 17, 2017. Spending most of his career in the Hadassah Medical Center in Jerusalem, Eliezer was one of the pioneers of thalassemia research, and one of the first to recognize the relationship between oxidative stress and iron overload in a variety of hematological disorders. He had a passion for medical research and until the last moments of his life he shared it with colleagues and students. He always showed an incredible learning ability and enthusiasm for new developments in basic and clinical research. Eliezer had an outgoing personality with a flair for approaching and attracting people and offering his professional assistance to those in need. He was endowed with a special skill for liaising between investigators specializing in different areas of biomedicine and, as such, he generated fruitful collaborations with numerous people worldwide, including those in Israel, the Palestinian territories, and Arab countries. Eliezer was the son of one of the legendary scholars and physicians who founded the Hadassah Medical Center in Israel as both and academic and clinical institution of worldwide reputation, and he continued that tradition of excellence until very recently. Eliezer will undoubtedly be missed by his family and by the extended biomedical community.

Gabriela Link , Prem Ponka, Abraham M Konijn, William Breuer, Z Ioav Cabantchik and Chaim Hershko . Department of Human Nutrition and Metabolism, Hebrew University Hadassah Medical School, Department of Biological Chemistry, Institute of Life Sciences, and Department of Medicine, Shaare Zedek Medical Center, Hebrew University of Jerusalem , Jerusalem, Israel and Lady Davis Institute for Medical Research, Montreal, Quebec, Canada

Abstract Background Ascorbic acid (AA) supplementation has traditionally been used in iron overloaded patients as means to increase iron chelation efficacy and replenish AA oxidized by labile iron found in those patients. The rationale leaned on AA's ability to render stored iron accessible to chelation, as found in urinary iron excretion following deferoxamine infusion. However, as AA increases labile iron redox-cycling and ensuing toxicity, we set to assess the long term benefits versus toxicity risks of the combined chelator-AA treatment. Objectives Perform a prospective, open-label, randomized and controlled 1 year study on thalassemia patients treated with deferasirox (DFX) in order to assess the effects of AA supplementation on: a. markers of systemic iron overload in selected organs and in plasma and b. markers of plasma labile iron (LPI) as potential contributors to oxidative stress toxicity. Patients and Methods Enrolment: 22 beta thalassemia major (TM) patients ≥10 years treated >2 years with DFX. Exclusion: cardiac dysfunction/arrhythmia or mT2* MRI <6 ms. Study: patients previously unexposed to AA received once-daily DFX (up to 40 mg/kg/d) with or without 125 mg AA for 1 year. All parameters were measured at baseline (BL); serum ferritin (SF) monthly, liver iron (LIC by MRI) and cardiac iron (mT2*MRI) after 1y. e-LPI (surrogate NTBI marker) and LPI (plasma redox-active labile iron marker) were assessed at BL, mo 1 & 6 by FeROS™ (Aferrix, Ltd) and fasting plasma AA at BL and EOS (fluorimetrically). Blood samples were withdrawn on the morning of transfusion day, 24 hours after last DFX (+/- AA) administration. Safety was followed using laboratory and clinical tests. AA levels were also determined in 23 healthy individuals (age and gender matched). Results 22 TM patients were enrolled (mean age 23.5, range 10-34 y). The average dose ± SD of DFX given to all 22 patients was 38±4.5 mg/kg/d. 11 patients were randomised to receive DFX and the others with DFX supplemented with 125 mg AA (mean 2.4±0.5, range 1.9-4.2 mg/kg) for 1 year. At BL, the AA levels were significantly lower in the TM group compared to controls (2.44 ± 3.38 vs 9.60± 4.36 mg/dl respectively, p<0.000001). 11 of 22 patients had AA levels >-2SD of control group whereas the other 11 patients showed normal ranges of AA. The AA deficient patients were those that showed significantly higher SF, LIC and lower mT2* at BL (Table 1). In the DFX+AA arm, 5/11 (45%) patients had subnormal AA levels at BL but attained normal status after 1 year, as did all others on AA. Of the 5/11 (45%) DFX-treated patients that did not receive AA had normal BL AA and only 2/11 maintained normal AA status at EOS. A significant correlation was obtained between BL SF, LIC and mT2* and e-LPI (r 0.49, p 0.025; r 0.57, p 0.01; r -0.43, p 0.057 respectively) but not with LPI. The changes associated with DFX alone or with AA from BL to EOS were subtle for all parameters measured (Table 2). Importantly, eLPI and LPI remained at basal levels throughout 6 months treatment in both arms. With DFX alone, LPI were 0.34±0.30 units (mM iron) (BL) & 0.63±0.58 (6 mo); eLPI: 1.71±1.93 at BL & 2.48±3.11 (6 mo). DFX+AA: LPI were 0.33±0.46 (BL) & 0.35±0.44 (6 mo); eLPI: 2.13±1.71 (BL) & 1.78±1.51 (6 mo). Conclusions TM patients on long term DFX without AA supplementation showed subnormal, AA levels. This was most pronounced in TM patients with higher liver and heart iron. The addition of AA to DFX normalized the AA levels but did not increase the e-LPI and LPI during 6 mo, indicating no apparent risk of iatrogenic toxicity by AA to DFX. Moreover, AA may enhance the efficacy of DFX in cardiac and hepatic iron. The small rise in SF versus fall in LIC in the DFX+AA arm might need further exploration. Table 1 Baseline characteristics of patients based on AA status Table 1. Baseline characteristics of patients based on AA status Table 2 Changes in iron overload markers in patients treated with DFX or DFX+AA over 1 year Table 2. Changes in iron overload markers in patients treated with DFX or DFX+AA over 1 year Disclosures Aydinok: Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cerus: Research Funding; Shire: Research Funding. Cabantchik:Aferrix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Hinoman: Consultancy; Novartis Pharmeceuticals: Honoraria, Speakers Bureau; Apopharma: Honoraria, Speakers Bureau.

CONTEXT:Type 2 Wolfram syndrome (T2-WFS) is a neuronal and β-cell degenerative disorder caused by mutations in the CISD2 gene. The mechanisms underlying β-cell dysfunction in T2-WFS are not known, and treatments that effectively improve diabetes in this context are lacking.OBJECTIVE:Unraveling the mechanisms of β-cell dysfunction in T2-WFS and the effects of treatment with GLP-1 receptor agonist (GLP-1-RA).DESIGN AND SETTING:A case report and in vitro mechanistic studies.PATIENT AND METHODS:We treated an insulin-dependent T2-WFS patient with the GLP-1-RA exenatide for 9 weeks. An iv glucose/glucagon/arginine stimulation test was performed off-drug before and after intervention. We generated a cellular model of T2-WFS by shRNA knockdown of CISD2 (nutrient-deprivation autophagy factor-1 [NAF-1]) in rat insulinoma cells and studied the mechanisms of β-cell dysfunction and the effects of GLP-1-RA.RESULTS:Treatment with exenatide resulted in a 70% reduction in daily insulin dose with improved glycemic control, as well as an off-drug 7-fold increase in maximal insulin secretion. NAF-1 repression in INS-1 cells decreased insulin content and glucose-stimulated insulin secretion, while maintaining the response to cAMP, and enhanced the accumulation of labile iron and reactive oxygen species in mitochondria. Remarkably, treatment with GLP-1-RA and/or the iron chelator deferiprone reversed these defects.CONCLUSION:NAF-1 deficiency leads to mitochondrial labile iron accumulation and oxidative stress, which may contribute to β-cell dysfunction in T2-WFS. Treatment with GLP-1-RA and/or iron chelation improves mitochondrial function and restores β-cell function. Treatment with GLP-1-RA, probably aided by iron chelation, should be considered in WFS and other forms of diabetes associated with iron dysregulation.

The centenary of Paul Ehrlich's death was commemorated by scientific communities worldwide, including at the 2015 International BioIron Meeting held by the International BioIron Society in Hangzhou, China (http://www.bioiron.org/). Chemotherapy, founded by Ehrlich (1854–1915; 1908 Nobel laureate), received particular recognition in 2015 by the Nobel Prize in Medicine being awarded to Tu Youyou for leading the team that introduced the antimalarial artemisinin (qinghaosu). (The 2015 Nobel Prize in Medicine was also awarded jointly to Satushi Omura and William Campbell for the discovery of novel antihelminth antibiotics. These scientists followed drug-screening methods that had led earlier to the discovery of streptomycin and 17 other antibiotics by Selman A. Vaksman (1952 Nobel Prize), largely based on work schemes applied by Paul Ehrlich.) Notably, although over a century has elapsed, the approach to medical research introduced by Ehrlich is still rendering novel therapeutic tools. The concept of targeted chemotherapy, whereby a chemical agent designed by nature or made in the laboratory acts as a magic bullet (zauberkugel) against a specific pathogenic target, is attributed to Ehrlich [[1]Witkop B. Paul Ehrlich and his magic bullets, revisited.Proc Am Phil Soc. 1999; 143: 540-547PubMed Google Scholar]. He arrived at this idea by studying cells stained with dyes. It was with a variety of staining techniques and careful examination that Ehrlich revealed the potential of vital stains such as methylene blue and Trypan blue as tools to identify features associated with individual blood cell types [[2]Buchwalow I. Boecker W. Tiemann M. The contribution of Paul Ehrlich to histochemistry: a tribute on the occasion of the centenary of his death.Virchows Arch. 2015; 466: 111-116Crossref PubMed Scopus (14) Google Scholar]. Ehrlich then extended the idea of chemical staining of a specific tissue/cell into a chemical tool for targeting a pathological agent. His maxim Corpora non agunt nisi fixata (compounds will not act unless they bind) reflected his rationale for designing organotropic and parasitotropic chemicals as anti-infective agents. Those agents became the tools of his Therapia magna sterilisans as they were selected for their ability to affect the pathogen while sparing the host (ideally with a single dose). By experimenting with methylene blue, he discovered its antimalarial potential as well as its analgesic power. Those observations paved the road for introducing atebrin as a quinine substitute and chlorpromazine for the management of severe neuroses and psychoses. The initial studies with chemical dyes as potential therapeutics in animal models of infection were subsequently expanded to derivatives of the pentavalent arsenic atoxyl, a chemical previously shown to be toxic to parasites but also to humans. In collaboration with Alfred Bertheim he improved the therapeutic index of atoxyl by systematic modification and testing in trypanosome-infected animals. In collaboration with Sashiro Hata he extended the tests to an animal model of syphilis (Treponema pallidum), which culminated with the generation of arsphenamine (compound 608), the first synthetic anti-infective that spared the host. Following extensive animal and human testing, tens of thousands of arsphenamine samples (commercialized as Salvarsan®) were initially offered freely to doctors worldwide for clinical assessment. Within a few years Salvarsan was replaced by the more easily dispensable, clinically effective and less toxic analogue Neosalvarsan® (No. 914). The ultimate goal of Therapia magna sterilisans became a reality in Ehrlich's lifetime, as with a single Salvarsan delivery, patients with frambesia (yaws) were cured from Spirochaeta pertenuis infection. Moreover, his view of using a combination of drugs with diverse chemical structures to overcome pathogen resistance and minimize the toxic effects of single overdosing drugs, gained direct support by treating African trypanosomiasis with arsphenamine in combination with acriflavine and ochlororosaniline. Although Salvarsan gradually ceased to be used after the advent of penicillin, Ehrlich's approach to drug discovery became a pre-eminent model for future generations, as exemplified in the discovery of the antibacterials sulphonamide (Prontosil) by Gerhard Domagk and streptomycin by Selman Vaksman, and as recognized by this year’s Nobel Prizes for the work on antiparasitic drugs. The search for antimalarial magic bullets, including synthetic analogues of quinine and antifolates, comprised large screening studies sponsored by the US and UK governments, WHO and by private foundations, leading to a small number of new drugs such as atovaquone and proguanyl, for which parasite multi-drug resistance rapidly emerged and spread across continents [[3]White N.J. Antimalarial drug resistance.J Clin Inv. 2004; 113: 1084-1092Crossref PubMed Scopus (841) Google Scholar]. Antimalarial drug combinations proved beneficial, particularly when compliance/adherence to treatment were strictly implemented. However, resistance to novel agents continued to pose a major challenge [[3]White N.J. Antimalarial drug resistance.J Clin Inv. 2004; 113: 1084-1092Crossref PubMed Scopus (841) Google Scholar]. During the North–South Vietnam war, China set up a top-secret national military project (No. 453) aimed at identifying antimalarial drugs among Chinese traditional herbal medicines [[4]Cui L. Su X.Z. Discovery, mechanisms of action and combination therapy of artemisinin.Expert Rev Anti Infect Ther. 2009; 7: 999-1013Crossref PubMed Google Scholar]. Trained as a phytochemist and in traditional medicines, Tu Youyou assembled a team that screened thousands of boiled water extracts from dried plants on Plasmodium berghei-infected mice. Partial but inconsistent success with extracts from qinghao (Artemisia annua) led Tu Youyou to search in the Handbook of Prescriptions for Emergencies (Ge Hon, 284–363 ad) for clues that led to the successful extraction of the active component of qinghao. Cold extraction, originally with water but currently with ether, reproducibly generated active parasiticidal mixtures that were more potent than chloroquine, enabling collaborating groups in the Yunnan Institute of Materia Medica to isolate, crystallize and chemically characterize the active agent (qinghaosu—artemisinin) and in the Guangzhou University of Traditional Medicine to demonstrate its clinical efficacy (against Plasmodium falciparum and Plasmodium vivax) and adequate safety. The road was opened not only for mass production of the drug but also for the search for more potent analogues and for strategies to overcome recrudescence associated with monotherapy [[3]White N.J. Antimalarial drug resistance.J Clin Inv. 2004; 113: 1084-1092Crossref PubMed Scopus (841) Google Scholar]. These have largely been achieved by artemisinin-based combination therapy, leading to improved pharmacokinetics, fast and long-lasting antimalarial efficacy and acceptable tolerability [[4]Cui L. Su X.Z. Discovery, mechanisms of action and combination therapy of artemisinin.Expert Rev Anti Infect Ther. 2009; 7: 999-1013Crossref PubMed Google Scholar]. From the outset, the antimalarial action of artemisinins has been attributed to the pro-oxidant endoperoxide moiety capable of generating toxic reactive oxygen species by interacting with labile iron in heme or other complexes assumed to be generated in parasitized cells [[4]Cui L. Su X.Z. Discovery, mechanisms of action and combination therapy of artemisinin.Expert Rev Anti Infect Ther. 2009; 7: 999-1013Crossref PubMed Google Scholar]. This concept might explain the specific (‘magic bullet’) effect of artemisinin on infected erythrocytes and the antagonistic effects afforded by iron chelators on its antimalarial activity—although the structural resemblance between thapsigargin and artemisinin has led some investigators to suggest that artemisinin affects parasite calcium homeostasis [[4]Cui L. Su X.Z. Discovery, mechanisms of action and combination therapy of artemisinin.Expert Rev Anti Infect Ther. 2009; 7: 999-1013Crossref PubMed Google Scholar]. Artemisinin has provided enormous benefit for public health. However, Plasmodium has developed resistance to its effects and, alarmingly, resistant forms of the parasite are spreading [3White N.J. Antimalarial drug resistance.J Clin Inv. 2004; 113: 1084-1092Crossref PubMed Scopus (841) Google Scholar, 5Ashley E.A. Dhorda M. Fairhurst R.M. Amaratunga C. Lim P. Suon S. et al.Spread of artemisinin resistance in Plasmodium falciparum malaria.N Engl J Med. 2014; 371: 411-423Crossref PubMed Scopus (1442) Google Scholar]. New antimalarials are likely to be needed, and in this search to discover chemotherapeutic agents, the scientific community should bear in mind Paul Ehrlich's famous maxim about the key elements of research success known as the four Gs: Geld (money), Geduld (patience), Geschick (skill) and Glück (luck) [[1]Witkop B. Paul Ehrlich and his magic bullets, revisited.Proc Am Phil Soc. 1999; 143: 540-547PubMed Google Scholar]. All these elements contributed to the discovery of artemisinin and arsphenamine, and it is most probable that they will be needed again to develop new ‘magic bullets’ against malaria and other diseases.