In the last 20 years, several modalities of neuromodulation, mainly based on non-invasive brain stimulation (NIBS) techniques, have been tested as a non-pharmacological therapeutic approach to slow disease progression in amyotrophic lateral sclerosis (ALS). In both sporadic and familial ALS cases, neurophysiological studies point to motor cortical hyperexcitability as a possible priming factor in neurodegeneration, likely related to dysfunction of both excitatory and inhibitory mechanisms. A trans-synaptic anterograde mechanism of excitotoxicity is thus postulated, causing upper and lower motor neuron degeneration. Specifically, motor neuron hyperexcitability and hyperactivity are attributed to intrinsic cell abnormalities related to altered ion homeostasis and to impaired glutamate and gamma aminobutyric acid gamma-aminobutyric acid (GABA) signaling. Several neuropathological mechanisms support excitatory and synaptic dysfunction in ALS; additionally, hyperexcitability seems to drive DNA-binding protein 43-kDA (TDP-43) pathology, through the upregulation of unusual isoforms directly contributing to ASL pathophysiology. Corticospinal excitability can be suppressed or enhanced using NIBS techniques, namely, repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS), as well as invasive brain and spinal stimulation. Experimental evidence supports the hypothesis that the after-effects of NIBS are mediated by long-term potentiation (LTP)-/long-term depression (LTD)-like mechanisms of modulation of synaptic activity, with different biological and physiological mechanisms underlying the effects of tDCS and rTMS and, possibly, of different rTMS protocols. This potential has led to several small trials testing different stimulation interventions to antagonize excitotoxicity in ALS. Overall, these studies suggest a possible efficacy of neuromodulation in determining a slight reduction of disease progression, related to the type, duration, and frequency of treatment, but current evidence remains preliminary. Main limitations are the small number and heterogeneity of recruited patients, the limited “dosage” of brain stimulation that can be delivered in the hospital setting, the lack of a sufficient knowledge on the excitatory and inhibitory mechanisms targeted by specific stimulation interventions, and the persistent uncertainty on the key pathophysiological processes leading to motor neuron loss. The present review article provides an update on the state of the art of neuromodulation in ALS and a critical appraisal of the rationale for the application/optimization of brain stimulation interventions, in the light of their interaction with ALS pathophysiological mechanisms. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the cortical, brainstem, and spinal motor neurons, leading to death due to respiratory failure usually within 3–5 years of symptom onset. While about 90% of cases are sporadic, the remaining are familial and usually inherited as dominant traits. Both familial and sporadic cases have been associated with mutations of several genes regulating protein homeostasis, RNA homeostasis, and cytoskeletal dynamics, with common downstream neuropathological and phenotypical alterations .Many studies, investigating neurophysiological and neuropathological alterations in both familial and sporadic ALS, converge on a “dying-forward” hypothesis at the origin of the disease, with cortical hyperexcitability as a possible driver of neurodegeneration. In this context, different molecular alterations of glutamatergic and gamma aminobutyric acid (GABA)-ergic neurons and receptors and of astrocytes, as well as the excitation/inhibition imbalance of motor cortical circuits, might all contribute the final picture of corticospinal hyperexcitability. These alterations suggest the possibility of exploiting therapeutic strategies pointing to hyperexcitability, targeted to specific molecular and/or functional alterations. Such a therapeutic approach is further valued by recent evidence that early cortical hyperexcitability is sufficient to trigger molecular alterations commonly associated with ALS , possibl+ channels, and it was shown to determine an increase of ~3 months in median survival time . Both drugs show modest effects on disease progression . Riluzolval time –6. Edaraval time . Howeverval time .Starting from 2004, different non-invasive brain stimulation (NIBS) techniques have been employed to modulate motor cortical excitability in the attempt to counteract excitotoxicity. The main attempts have been made with repetitive transcranial magnetic stimulation (rTMS), allowing repeated activation of cortical synapses, and with transcranial direct current stimulation (tDCS), acting on resting membrane potential: both techniques can produce a prolonged suppression of neuronal excitability through modulation of synaptic plasticity in physiological conditions . AdditioIn this review, we provide an update on brain stimulation interventions applied in ALS and on their potential benefits and current limitations, in the light of their interaction with mechanisms of corticospinal excitability.A full knowledge of ALS pathogenesis is still to be uncovered; however, the known pathogenic mechanisms and resulting dysfunctions are fundamental in developing targeted therapeutic interventions.The two main histological characteristics of the disease, described by the terms “amyotrophic” and “lateral sclerosis,” derive from the degeneration of motor neurons in the anterior horn of the spinal cord and from axonal loss in the lateral columns , respectively.After the initial Charcot's hypothesis on the primacy of corticomotoneuronal dysfunction , the so-In the last three decades, accumulating evidence supports the initial hypothesis of the hyperexcitability of the corticomotoneuronal system as a leading pathogenic mechanism. This view is supported by several elements: (1) prominent muscle wasting in districts with direct corticospinal projections and sparing of LMN not controlled by corticospinal tract ; (2) time-dependent spreading of neuropathological alterations from layer V pyramidal cells to structures under direct control of corticofugal projections; (3) clinical syndromes characterized by frontal dysfunction preceding muscle weakness; (4) neurophysiological evidence of hyperexcitable corticospinal circuits; (5) cortical hyperexcitability documented before LMN dysfunction; and (6) evidence of altered glutamatergic signaling [reviewed in Eisen et al. ].Based on the above considerations, it is assumed that hyperexcitability represents a common pathogenic mechanism leading to excitotoxicity and degeneration of upper and lower motor neurons. Hyperexcitability can be mediated either by direct pre-synaptic mechanisms , due to increased glutamate stimulation, or by indirect post-synaptic mechanisms , due to altered interneuron regulation, glutamate receptors, or intrinsic motoneuronal excitability .The link between UMN hyperexcitability and LMN degeneration remains a matter of debate, and a mechanism of glutamate-mediated excitotoxicity from the direct corticospinal connection is not demonstrated. Indeed, findings on alterations of spinal circuit excitability in ALS are not conclusive, as different pathways and interneurons characterize animal models and humans. We refer the reader to existing literature for details on the complexity of spinal circuit pathophysiology  at the time of neurophysiological investigation, reports that cortical hyperexcitability increases with disease duration, excluding subjects with inexcitable M1 to TMS  point to the occurrence of phenomena of concomitant increased CSN excitability and of reduced intracortical inhibitory activity . Interes1 to TMS . Of note1 to TMS , 32.It remains an open question whether hyperexcitability represents, at least in part, a compensatory phenomenon of motor neuron degeneration . While iGrowing body of evidence also reveals that ALS exhibits subclinical peripheral and central sensory neuron dysfunctions –39. IndeDespite the entire picture of pathophysiological mechanisms of hyperexcitability in ALS remains to be uncovered, the known physiological alterations support the rationale of interventions based on reducing corticospinal activity aimed at preventing upper and/or lower motor neuron excitotoxicity.Although specific mechanisms underlying selective degeneration of motor neurons in ALS remain unknown, some neuropathological alterations might underlie degeneration, abnormal cell excitability, and synaptic transmission and thus account for cortical circuit dysfunction. Conversely, neuronal hyperactivity can lead to molecular alterations accounting for cell dysfunction and degeneration.Genetic studies have identified a high number of genes associated with both familial and sporadic ALS; however, both forms show a substantial convergence in their pathological features leading to motor neuron degeneration .The neuropathological hallmark of the disease is the presence of transactivation response DNA-binding protein 43-kDA (TDP-43) positive inclusions into neuronal cytoplasm  frequentper se can promote the expression of proapoptotic genes. In mouse models, cytoplasmic mislocalization of TDP-43 induces apoptosis even in the absence of aggregates. In addition, aberrant TDP-43 alters splicing and RNA metabolism. Finally, aggregates of hyperphosphorylated and fragmented TDP-43 protein are associated with stress responses and facilitate cell death -positive neurons, providing inhibitory input to SST+ cells, and VIP-negative neurons, connected to the soma or dendrites of pyramidal cells through GABAA or GABAB receptors  and anodal (facilitatory) tDCS, anodal tDCS also showed facilitation of the I1 wave  and of tMoreover, a comparison of descending volleys evoked by transcranial and epidural motor cortex stimulation indicates that epidural stimulation can produce repetitive excitation of CSNs similar to TMS , supportIt must be considered that the effects of modulatory interventions on corticospinal activity were measured in a small number of subjects: this limits the level of certainty of proposed models of interaction of different brain stimulation protocols with motor cortical circuits. However, based on current evidence, we can expect that certain types of interventions suppressing M1 excitability, such as cTBS, will mainly affect excitatory intracortical inputs to layer V pyramidal cells, while other protocols, such as 1-Hz rTMS, PAS, and cathodal tDCS, will affect more complex intracortical circuits including GABAergic interneurons connected to layer V pyramidal cells. Moreover, based on the biophysical properties of the technique and on very preliminary data, it is conceivable that tDCS has also some direct modulatory effect on CSN excitability in addition to its interaction with synaptic plasticity , 86.A systematic search for original studies applying brain or spinal stimulation on ALS subjects, with the purpose of persistent neuromodulation aimed at affecting functional status, was performed on 10th August 2020 in the PubMed database , using the following search terms:#1 [als amyotrophic lateral sclerosis (MeSH Terms)] OR [motor neuron disease (MeSH Terms)] OR [amyotrophic lateral sclerosis (Title/Abstract)] OR [ALS (Title/Abstract)];#2 [transcranial magnetic stimulation (MeSH Terms)];#3 [electrical stimulation of the brain (MeSH Terms)];#4 [magnetic field therapy (MeSH Terms)];#5 [stimulation (Title/Abstract)];#6 {[magnetic (Title/Abstract)] AND [repetitive (Title/Abstract)]} OR [current (Title/Abstract)] OR [electrical (Title/Abstract)] OR [epidural (Title/Abstract)] OR [spinal (Title/Abstract)] OR [focused ultrasound (Title/Abstract)] OR [static magnetic field (Title/Abstract)];#7 [TMS (Title/Abstract)] OR [tDCS (Title/Abstract)] OR [tACS (Title/Abstract)]Combined query: #1 AND [#2 OR #3 OR #4 OR (#5 AND #6) OR #7].Query produced 431 results, which were screened on the basis of title and abstract; 28 remaining full-text articles were assessed for eligibility; 14 original studies were included in qualitative synthesis  rTMS in two subjects and high-frequency (20 Hz) rTMS in two subjects : the ratAnother pilot controlled trial tested 5-Hz rTMS, delivered to M1 bilaterally for 2 weeks, in five ALS subjects . The ratNon-invasive techniques using electrical stimulation encompass tDCS and transcutaneous spinal stimulation. At present, other techniques of brain stimulation using a variable current intensity, such as transcranial alternating current stimulation (tACS), have not been employed in therapeutic trials in ALS.In the last decade, tDCS was tested mainly in the attempt of obtaining a portable therapeutic option that does not require to be delivered within the hospital setting. Indeed, tDCS requires only a small size stimulator and it can be delivered even at patients' home.To date, the number and size of tDCS studies is overall even smaller than that of rTMS studies. In the only study  in whichFrom a physiological point of view, Munneke et al.  had failClinicalTrials.gov ID: NCT04293484).Recently, Benussi et al.  tested aClinicalTrials.gov ID: NCT03411863).A recent feasibility neurophysiological study  also conInvasive cortical or spinal stimulation might represent an interesting translational prospect of preliminary findings obtained non-invasively into therapeutic strategies based on increased stimulation dosage. However, to date, only very few cases of ALS patients with implanted stimulators have been reported.Cortical subdural stimulation was initially tested by Sidoti and Agrillo  in four The effects of epidural spinal stimulation in ALS have been investigated by Di Lazzaro et al.  in one sThe studies analyzed above, including a total of <100 ALS patients undergoing active treatments with either rTMS, tDCS, or invasive subdural/epidural stimulation, did not report any adverse event. Mild dysesthesias under tDCS electrodes might be perceived . It mustSeizures are the most feared adverse event with non-invasive brain stimulation, although they are a rare event. No seizures have been reported in the above-analyzed studies with ALS patients. Inhibitory rTMS and tDCS protocols are traditionally considered at lower risk of inducing seizures. A recent analysis on more than 300,000 TMS sessions  indicateFinally, due to the relatively short time of observation and the small number of recruited patients in ALS trials, long-term effects of prolonged brain stimulation procedures could not be investigated.Measuring the effects of new therapeutic interventions in ALS is challenging. Besides survival, requiring large samples, and long trials, more selective indices of disease course are usually employed to increase sensitivity in small trials. However, distinct measures have a different sensitivity in catching specific functional and pathophysiological alterations that can be differentially affected during disease progression.In this section, we summarize the main outcome measures that can be used to assess the response to neuroprotective interventions based on brain stimulation techniques, ranging from functional clinical scales measuring the overall level of disability to neurophysiological tools assessing specific dysfunctional mechanisms implicated in ALS pathophysiology .Initial attempts for developing a functional rating scale were pursued in the 1970s, namely, the Norris Scale and its modified version that consists of two parts, i.e., the Limb Norris Score, composed of 21 items, and the Norris Bulbar Score, composed of 13 items . HoweverALSFRS-R is largely used in several clinical trials, it is easy to perform remotely for telehealth, and its self-administered version shows good reliability and sensitivity to change over time vs. the standard evaluator-administered ALSFRS-R . HoweverImportantly, over the last few years, two functional scales [the King's College and Milano-Torino Staging (MiToS) staging systems] have been proposed to staging ALS. The King's College system uses five stages, ranging from 1 being symptom onset and stage 5 being death, and relies on the clinical spread of disease among the several clinical regions as a measure of progression . InsteadLoss of muscle strength is a cardinal feature of ALS. Functional loss over time, including respiratory dysfunction, inability to ambulate, loss of ability to perform activities of daily living, and others are due, in large part, to decline in strength. Therefore, the accurate measurement of limb muscle strength and respiratory function is essential in therapeutic trials to better understand the impact of therapy on vital function.With regards to limb muscle strength evaluation, we have a qualitative assessment with the manual muscle testing (MMT) scale established by the Medical Research Council of the Royal College of Physicians and Surgeons (MRC), scoring from 0  to 5  each tested muscle. However, the measure itself is subjective; therefore, to overcome such limitations, quantitative scales have been developed , and with careful training and reliability testing, they have proven to be reliable and sensitive indicators of disease progression. The ability to determine objectively the source of weakness would be of great value and should be a subject of future research .Respiratory failure is the primary cause of death in ALS. Interestingly, approximately in 3–5% of cases, ALS can begin with respiratory failure –128. ChaAccording to American  and EuroAlthough motor system deficits may appear prominent, ALS is increasingly recognized as a multisystem disorder accompanied by cognitive changes .In contrast to the relentlessly progressive motor deficits, the trajectory of cognitive and behavioral deficits is less clear due to considerable individual variations, genotype-associated profiles , 135, diCognitive and behavioral domains are routinely assessed thanks to validated screening instruments such as the Edinburgh Cognitive and Behavioral ALS Screen (ECAS) , the BeaSince ALS is characterized by progression to death, survival is recommended as the primary endpoint for phase 3 trials . Indeed,Survival analysis, in its wider conception, encompasses approaches to investigate the time for an event of interest to occur and is largely based on Kaplan–Meier estimates. Since the time to death in ALS is extended by nutritional and respiratory interventions, some trials included time to tracheostomy or prolonged non-invasive ventilation as part of survival outcome, but the use of respiratory interventions differs between centers and it could even increase variability of these combined measures .Considering that survival time in ALS is quite variable, in order to reach a sufficient statistical power, the two fundamental requirements are that (a) the time of observation must be long enough relative to the average disease course and (b) the groups must be large enough to overcome intrinsic variability and appreciate differences. Survival analysis is also biased by censored patients, i.e., those subjects who are missed at follow-up for any reason and exit the population “at risk,” thus flattening the end of the survival curve. If the number of censored subjects is high, and this might well be the case in long-lasting ALS trials, it will affect the accuracy of survival estimation. Moreover, the above requirements are not satisfied by pilot studies, which usually aim to explore the possibility of a further investment on a new treatment strategy and therefore switch to functional or physiological outcome measures.Traditionally, survival and function have been assessed as independent endpoints in ALS trials. Combined approaches have been implemented to reduce the confounding effect of mortality on analysis of functional outcomes. Most notably, the Combined Assessment of Function and Survival (CAFS) calculates a rank for each subject, based on survival time and decline in the ALSFRS-R score: in this way, a mean rank score can be calculated that allows for statistical comparison between treatment groups . AdditioNeurophysiological techniques might provide an objective measure of UMN and LMN dysfunction in ALS. For this reason, they seem to be more sensitive to detect disease progression with respect to clinical scales. Neurophysiological abnormalities might also uncover UMN and LMN deficit that are clinically silent, thus enabling an earlier diagnosis and thereby recruitment into therapeutic trials. Additionally, they could provide important insights into disease mechanisms that ultimately lead to uncovering of novel therapeutic targets.Neurophysiological Insights section above).Single and paired-pulse TMS techniques, providing parameters such as MT, central motor conduction time (CMCT), cortical silent period, and intracortical inhibition and facilitation, have gained credibility as a clinical tool to investigate the integrity and excitability of the corticomotoneuronal system  amplitude, may be relatively insensitive in assessing LMN degeneration due to the process of reinnervation .The neurophysiological index (NI), using a simple formula, combines routine CMAP amplitude, F-wave frequency, and distal motor latency to improve sensitivity in demonstrating longitudinal LMN loss in ALS . NI is aRegarding the use of F-waves, cortical and peripheral mechanisms have been proposed to account for the F-wave abnormalities , 160. F-Motor unit number estimations techniques, such as motor unit number estimation (MUNE) and motor unit number index (MUNIX), may potentially represent valuable biomarkers of LMN degeneration. Studies utilizing various MUNE techniques in ALS patients and healthy controls have reported good intra-rater and inter-rater reliability –165. In + channel-blocking agents. Specifically, low-dose mexiletine (300 mg) did not exert any modulating effects on axonal excitability parameters, potentially accounting for the absence of clinical effectiveness . Moreover, an intrinsic limitation in this field relates to the fact that most rTMS studies were conducted by the same research group.Related to the previous point, it is a matter of great importance and complexity selecting suitable endpoints, to which we have dedicated a section of the present work. Indeed, distinct outcome measures have a different sensitivity in catching specific functional and pathophysiological alterations that can be differentially affected during the disease course. Trials with prolonged  interventions substantially used functional and strength scores as the main outcome measures, with the ALSFRS-R almost constantly employed. These outcome measures are subject to high variability among individuals and along disease progression. The existence of even infrequent “plateaux” and “reversals” in the ALSFRS score decline  further A second important limitation in ALS trials is the heterogeneity of recruited patients. Inclusion of subjects with different phenotypes and/or disease stages considerably increases the within-group variability and required sample size. Recruitment difficulties, mainly related to the high effort required for patients to regularly reach clinical centers to undergo NIBS procedures and the difficulty to plan the exclusion of many potentially highly motivated subjects in the absence of other therapeutic options, could account for study protocols with wider inclusion criteria.The disease severity and duration at the time of enrolment also plays an important role in the evaluation of treatment outcome, for two main reasons. First, ALS progression, as evaluated by the ALSFRS score, typically slows in the more advanced stages  and thisDealing with the purpose of early recruitment is challenging since the sensitivity of the clinically based criteria is limited, particularly in the early stages of ALS –188, leaBesides limitations related to experimental design, the characteristics and neurobiological effects of brain stimulation interventions must be considered. We described how different NIBS protocols interact with corticospinal circuitry. Protocols targeting indirect intracortical connections with CSNs , such as 1-Hz rTMS, PAS, and cathodal tDCS, have the potential to interact with mechanisms of hyperexcitability related to hypoactive GABAergic interneurons, demonstrated by neurophysiological and neuropathological investigations. Otherwise, protocols targeting direct projections to CSNs or CSNs themselves, such as cTBS and possibly tDCS, could reduce intrinsic UMN excitability. However, based on current knowledge, it is not possible to foresee which of the above hypothetical mechanisms (upstream or direct interaction with CSNs) will be more efficient. This is substantially due to a still insufficient knowledge on the pathological cascade at single-cell level and on the exact interaction of brain stimulation techniques with individual cell populations. Moreover, we do not know to which extent counteracting corticospinal hyperactivity might prevent LMN degeneration , even ifStudies on ALS patients also do not provide sufficient indications on what might be the best stimulation paradigm. To date, cTBS was tested in the majority of patients, with some favorable results in the first months of treatment  and at hExperimental data also suggest that hyperexcitability in ALS might be a compensatory phenomenon of cell loss : in thisFinally, it must be accepted that, if most or the totality of ALS forms depend on gene alterations and if hyperexcitability is not a causal factor of neuropathological alterations and cell loss, all interventions targeted to counteracting hyperexcitability can be expected, at best, to slow the disease progression without substantial effects on long-term survival.ClinicalTrials.gov ID: NCT04393467). Alternatively, implanted devices could warrant prolonged daily stimulation, but their interaction with intracortical circuits is still largely unexplored.In any case, since the physiological effect of NIBS techniques on cortical excitability is usually short-lasting, the time during which the motor cortex is exposed to stimulation appears as a key factor to produce clinically significant effects. The application of rTMS protocols is limited by the necessity of being administered within the hospital, while tDCS could be implemented for home self-administration. The administration at the patient's site of a new NIBS technique, transcranial static magnetic field stimulation , 195, haIn conclusion, in our view, there are few approaches that could warrant informative results from future trials.Targeted patients' selection. Subjects in the early stages of disease, stratified based on clinical characteristics and biomarkers, could represent a population exhibiting less variability and more likely to benefit of the potential disease-modifying effect of brain stimulation interventions.Adequate sample size. Samples larger than those tested until now, which could be obtained with multicenter studies, will allow to perform phase 2 or phase 3 clinical trials once a promising intervention has been defined from pilot studies.Targeted stimulation protocols with appropriate outcome measures. Further research on the mechanisms of corticospinal dysfunction in ALS and on the way to target specific cortical or corticospinal circuits will allow to target specific pathophysiological mechanisms. Neurophysiological or molecular biomarkers could allow to detect biological effects of proposed treatments before their clinical translation.Ensuring suitable stimulation dosage. Based on the current knowledge on the time course of neurophysiological effects of most brain stimulation techniques, it appears that protocols tested until now are likely underdosed to produce clinically significant effects. Increased dosage can be ensured with either repeated sessions of self-administered non-invasive stimulation or continuous stimulation delivered through implanted devices. The best risk-to-benefit ratio for different protocols remains to be determined.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors VD.


This study is a systematic literature review of cost analyses conducted within implementation studies on behavioral health services. Cost analysis of implementing evidence-based practices (EBP) has become important within implementation science and is critical for bridging the research to practice gap to improve access to quality healthcare services. Costing studies in this area are rare but necessary since cost can be a barrier to implementation and sustainment of EBP.We followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology and applied the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist. Key search terms included: (1) economics, (2) implementation, (3) EBP, and (4) behavioral health. Terms were searched within article title and abstracts in: EconLit, SocINDEX, Medline, and PsychINFO. A total of 464 abstracts were screened independently by two authors and reduced to 37 articles using inclusion and exclusion criteria. After a full-text review, 18 articles were included.Findings were used to classify costs into direct implementation, direct services, and indirect implementation. While all studies included phases of implementation as part of their design, only five studies examined resources across multiple phases of an implementation framework. Most studies reported direct service costs associated with adopting a new practice, usually summarized as total EBP cost, cost per client, cost per clinician, and/or cost per agency. For studies with detailed analysis, there were eleven direct cost categories represented. For five studies that reported costs per child served, direct implementation costs varied from $886 to $9470 per child, while indirect implementation costs ranged from $897 to $3805 per child.This is the first systematic literature review to examine costs of implementing EBP in behavioral healthcare settings. Since 2000, 18 studies were identified that included a cost analysis. Given a wide variation in the study designs and economic methods, comparison across studies was challenging, which is a major limitation in the field, as it becomes difficult to replicate studies or to estimate future costs to inform policy decisions related to budgeting. We recommend future economic implementation studies to consider standard economic costing methods capturing costs across implementation framework phases to support comparisons and replicability. Contributions to the literatureImplementation research of evidence-based behavioral health interventions has grown dramatically in the past 10 years; however, costing methods and types are still not standardized which makes cost comparisons across studies difficult.We describe the types of costs and costing methods which have been used to date in implementation research of evidence-based behavioral health interventions.Results of this analysis inform future studies in identifying appropriate costs and methods to include in research which will improve rigor and replicability.Cost analysis is broadly applied through economic evaluations of treatment interventions and related programs for mental health/substance use disorders (SUD), often as part of an effectiveness/cost-effectiveness study. Recently, cost analysis has become an important area within the implementation science field, which focuses on the translation of research into practice and disseminating methods and applications of evidence-based practices (EBP) on a broader scale . ImplemeIn their seminal article, Proctor et al. specifically called for implementation research to include costs as an outcome . The incImplementation studies within the broader health service delivery field that included economic evaluations were summarized in a recent systematic literature review by Roberts et al. . This reThe extent to which researchers have included costs in their implementation studies of EBP in behavioral health services is not known. Additionally, the costing methods applied in these studies have not been systematically described. We address this gap by conducting a systematic review of the literature to understand the types of costs and costing methods which have been used to date in implementation research of evidence-based behavioral health interventions in the USA and Canada. We focus on these two countries due to their similarities in the epidemiology of behavioral health disorders, overlapping related health policies , and floWe conducted an initial systematic review (February 2019) and updated the search prior to submission (April 2020). We investigated costing studies in behavioral health implementation research using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology  and checWe based our search strategy on suggestions from psychology and economics published best practices for systematic literature reviews , 15. Ter((TI economic) OR (TI cost) OR (AB economic) OR (AB cost)) AND ((TI implementation) OR (AB implementation)) AND ((TI evidence based practice) OR (AB evidence based practice)) AND  OR  OR  OR  OR  OR  OR  OR  OR (TI psychiatric) OR (AB psychiatric) OR (TI addiction) OR (AB addiction) OR (TI drug abuse) OR (AB drug abuse) OR (TI substance use) OR (AB substance use) OR (TI drug use) OR (AB drug use))Articles that address implementation studies of behavioral health services which incorporate costing Studies published after 2000Studies only if original research is presented Community-based studies Peer-reviewed articlesPublished in EnglishResearch was conducted in the USA or CanadaStudies providing services to people of any ageThe following are the inclusion criteria:Published before 2000Editorials, newspapers, and other pieces of popular mediaDissertations or thesesBook chapters, book reviews, proceedings, government and/or organization reports, and other publications from the gray literatureSystematic reviewsProtocols without preliminary dataCase studyNon-implementation-based studiesPublished in a language other than English.Studying a system outside the USA or CanadaStudies based in hospitalsDid not include costing dataFocusing solely on comparing medicationsThe following are excluded in the study:As shown in Fig. The following characteristics were extracted from all included studies: authors, year published, journal, area of behavioral health, type of EBP, sample size, implementation framework, economic methodology, costing approach, costing perspective(s), cost categories and specific resources, cost analysis results, and economic study design. Thematic analysis was conducted to summarize similarities and differences across studies. Content analysis was used to identify how these characteristics overlapped within included studies, such as identifying costing perspectives and resource categories that overlapped across studies. Content analysis classified three categories of costs for the purpose of the study: direct implementation, direct service, and indirect implementation. For a subset of studies, direct and indirect implementation cost results were extracted and reported. In studies where summary cost estimates were not provided, we used the aggregated cost results (as presented) combined with patient/family/client sample sizes provided within the articles to approximate summary costs  to support comparisons across studies.The Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist  was usedTable Most articles (15 articles) investigated the implementation of a single EBP. However, some studies (three articles) considered implementation of multiple practices , 33, 36.Studies varied in sample size and unit of analysis. Four studies measured EBP adoption on individual patient outcomes. The sample size in these studies ranged from 72-1,869 people; however, three of the studies had a sample size between 72-155 , 25, 34.Most articles Most implementation frameworks follow a phased structure, generally including pre-implementation, implementation, and sustainment activities and corresponding evaluation metrics. Cost analyses must define an analytic perspective, and in implementation research, two key perspectives are the agency/provider/organization that would be deciding to adopt an EBP and the payer, sometimes the agency itself, health insurance plan, or Medicaid/Medicare. In cost-effectiveness research, the Second Panel on Cost-Effectiveness in Health and Medicine recommends adopting the healthcare sector and societal perspectives, both of which are broader than a provider or payer perspective. The societal perspective includes costs to the healthcare system, other systems , and patient/caregivers. The cost analysis perspective defines the relevant resources and monetary conversion factors for estimating costs across implementation phases. General categories of implementation costs include direct labor costs, indirect labor costs, and nonlabor costs . Direct For the purposes of this review, we classify three categories of costs guided by the presentation of costs in the included studies: direct implementation costs, direct service costs, and indirect implementation costs Table . As showIndirect implementation costs capture the opportunity cost for agencies due to lost revenues and/or time spent on implementation activities rather than standard (pre-implementation) clinical activities that could be billed and reimbursed. Indirect costs were estimated using the amount of time that a clinician reported spending on implementation activities, multiplied by the reimbursement rate for their billable activities , 28. TwoUsing the cost categories defined in Table As shown in Table Studies reporting quantitative cost results across the highlighted costing categories are summarized in Table Three studies examined costs across phases of implementation: pre-implementation, implementation, and sustainment. All three of these studies also reported average per-child costs. One study combined pre-implementation and implementation direct costs totaling $886 per child . A seconResults from the CHEERS checklist Table  indicateThis is the first systematic literature review to examine cost analyses within implementation studies of EBP for behavioral health services. The literature review identified 18 studies published since 2000 that included a formal cost analysis. Most of this work has been published in recent years (16 articles since 2013). Given the increase in cost analyses within implementation research of behavioral health EBPs, this study provides important context for the current state of the field, including a summary of findings, and we offer suggestions for how the field might standardize economic concepts and measures to increase translatability and support data harmonization going forward. In particular, the large number of not applicable categories from the CHEERS checklist may indicate a need for a new checklist of costing as part of implementation research.We found several studies proposing the development of an implementation research framework, but no studies using a previously published framework. This finding resonates with a recent study by Wensing and Grol (2019) that identified a large number of implementation frameworks within the field, but very little consistency across contextual determinants . Our finWe identified a wide variation in study type and costing methods, thus limiting the comparability across cost analysis results. Further complicating comparability is the possibility that there could be some double counting in the direct implementation and direct service cost categories if staff time is being reported as a provider expense as well as a billable service cost. Only one study included both of these categories , and theRegarding the presentation of summary cost estimates, three studies examined the same EBP , which facilitated comparison. However, even among these studies there was wide variation in reported costs, which may suggest that costs are substantially impacted by factors outside of the implementation process. Some of the reported costs were also relatively high , and may be inflated due to the inclusion of fixed startup costs  and smaller caseloads. In fact, one of the included studies highlights this in reporting that per child and per session incremental implementation costs are highest in the start-up phase and decrease over time as more children are served . HoweverGiven the wide variation in the types of factors included in implementation research it is difficult to make comparisons across studies. This is a major limitation of implementation research focusing on behavioral health services, as it becomes difficult to replicate studies or use studies to estimate future costs to inform policy decisions related to budgeting. With increasing emphasis on the economics of implementation science , the fieBased on our findings, we recommend that future studies include more details about the specific activities and resources associated with an implementation process, so that other researchers and policy makers can anticipate what costs will be incurred in changing existing or adopting new practices. We also recommend that studies include measures of environmental context such as treatment capacity, available funding (i.e.: block grants) and urbanicity, all available from public sources, to better capture how outside factors may impact the costs of implementing EBPs . As showWhile this study adds substantially to the literature by describing the state of the field to date, there are still several limitations. For example, since there was very little overlap between studies in the types of EBP being implemented or outcome measures, we were unable to conduct a meta-analysis. Our findings were also focused on implementation studies of evidence-based behavioral health services, and therefore do not extend to other health-related services. Further, only one included study reported both direct implementation costs and direct service costs , which lImplementation research on EBPs in behavioral health has grown significantly in the last several years. However, the field has not yet standardized the use of economic methods or measures. Recommendations based on our findings include moving toward standard cost measures to facilitate cross study comparisons and the potential for results to drive policy decisions.


Hepatitis B virus (HBV) is considered a “metabolic virus” and affects many hepatic metabolic pathways. However, how HBV affects lipid metabolism in hepatocytes remains uncertain yet. Accumulating clinical studies suggested that compared to non-HBV-infected controls, chronic HBV infection was associated with lower levels of serum total cholesterol and triglycerides and a lower prevalence of hepatic steatosis. In patients with chronic HBV infection, high ALT level, high body mass index, male gender, or old age was found to be positively correlated with hepatic steatosis. Furthermore, mechanisms of how HBV infection affected hepatic lipid metabolism had also been explored in a number of studies based on cell lines and mouse models. These results demonstrated that HBV replication or expression induced extensive and diverse changes in hepatic lipid metabolism, by not only activating expression of some critical lipogenesis and cholesterolgenesis-related proteins but also upregulating fatty acid oxidation and bile acid synthesis. Moreover, increasing studies found some potential targets to inhibit HBV replication or expression by decreasing or enhancing certain lipid metabolism-related proteins or metabolites. Therefore, in this article, we comprehensively reviewed these publications and revealed the connections between clinical observations and experimental findings to better understand the interaction between hepatic lipid metabolism and HBV infection. However, the available data are far from conclusive, and there is still a long way to go before clarifying the complex interaction between HBV infection and hepatic lipid metabolism. Hepadnaviridae family, is one of the smallest enveloped animal DNA viruses and the pathogen to cause Hepatitis B. Although HBV vaccines and effective antiviral drugs have been available for more than 20 years, chronic HBV infection (CHB) remains a global public health problem, especially in Asia (Hepatitis B virus (HBV), a member of the  in Asia . Increas in Asia , includi in Asia , farneso in Asia , estroge in Asia , or pero in Asia . Simulta in Asia . Therefo+) populations (38.9%) than in the HBsAg− group (44.5%), and HBsAg positivity was inversely correlated with fatty liver. These results were consistent with another large-sample prospective cohort study  and non-alcoholic fatty liver disease (NAFLD). So far, the prevalence of NAFLD in CHB patients remains unconfirmed. A meta-analysis conducted in rt study , which srt study . In addirt study . A studyrt study .Apart from the comparison of the prevalence of hepatic steatosis between CHB patients and non-CHB controls, factors involved in NAFLD development among CHB populations were thoroughly investigated in some studies. Factors that had positive or negative influence on hepatic steatosis in CHB patients were shown in Nevertheless, these clinical studies suggested that HBsAg positivity might be related to NAFLD’s low occurrence, while the status of liver inflammation and clinical or metabolic factors during disease progress would influence this relationship. Furthermore, differences in clinical study design, sample size, method for hepatic steatosis detection, or statistical method would deduce different conclusions. Clarifying the relationship between HBV infection and NAFLD development will require additional multi-center, large-sample, case-controlled clinical studies with hepatic steatosis assessment in liver biopsy samples.Fatty acid metabolism in the liver is controlled by a complex network of TFs and proteins . In diffLevels of various types of lipids were also examined by mass spectrometry in different stages of chronic HBV infection. Apart from reports that HBV increased lipogenesis, several other studies showed that HBV infection also increased fatty acid oxidation and reduced lipid droplets (LD) formation. Overall, these results from experimental studies indicated that HBV infection not only enhanced fatty acid synthesis and lipogenesis, but also increased fatty acid oxidation and lipolysis.+ patients compared to HBsAg− individuals. This study also found that phosphatidylcholine synthesis-related enzymes, phosphoethanolamine transferase A and phospholipid phosphatase 1 (LPP1), were upregulated after HBV infection. Different pattern of phospholipid changes was displayed in different stages of chronic HBV infection. In addition, significant changes in phospholipids and sphingolipids were found in HBV expressed primary hepatocytes or cell lines and CHB patients. Changes in cholesterol metabolism were evidenced during HBV infection. +/taurocholate cotransporter (NTCP/SLC10A1), a sodium-dependent transporter responsible for the basolateral uptake of taurocholate, was found as an entry receptor for HBV  and played a central role in the transport, metabolism, and homeostasis of cholesterol and other lipids . HBV infThese results indicated that HBV replication had complex effects on lipid metabolic pathways . HoweverAs reviewed above, HBV infection induced a series of changes in the hepatic lipid metabolic pathways. On the other hand, changes in the levels of metabolic products or expression of metabolism-related proteins conversely affected the life cycle of HBV .Cholesterol was necessary for the HBV envelope and played a critical role in HBV infectivity or viral particle secretion . CholestThe effect of bile acid on HBV infection has received increasing attention. Studies showed that high expression of NTCP induced an increase in bile acid transport to hepatocytes, which promoted HCV and HBV infection by bile-acid-mediated repression of some interferon-stimulated genes . Also, eHepatitis B virus infection induces multiple changes in hepatic lipid metabolism, by increasing both lipid synthesis and lipolysis. These intracellular changes might be helpful to HBV replication through providing both material and energy. These observations also elucidate why HBV infection facilitates lipid synthesis while the risk of hepatic steatosis in CHB patients does not noticeably increase, and levels of serum TG and TC decrease. Moreover, targeting a certain lipid metabolism pathway would be a potential therapeutic way to chronic hepatitis B. Given that many studies are limited to cells and animal models and evidence is far from conclusive, more well-designed clinical studies are needed to elucidate the mechanism of this interaction and to discover metabolic targets that could suppress HBV replication or improve the anti-HBV effect of IFN or nucleot(s)ide analogs.JZ, NL, and YL: conception and literature review. JZ and MC: writing of the original draft. MC and PH: review and editing of the manuscript. MC: funding acquisition. MP and PH: supervision of the manuscript. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Biological therapies are currently the mainstay in the treatment of patients with inflammatory bowel diseases (IBD). Several factors are known to influence the efficacy and tolerability of biologicals, such as CRP levels or previous biological use. Whether patient sex affects the efficacy or tolerability is unclear but would help with better risk and benefit stratification. This systematic review assesses patient sex on the efficacy and tolerability of biological therapies in IBD patients.A systematic literature review was performed using Embase (including MEDLINE), MEDLINE OvidSP, Cochrane Central Register of Controlled Trials, Web of Science and PubMed. The primary outcome was the influence of patient sex on endoscopic outcomes in IBD patients treated with biologicals. The secondary outcome was the influence of patient sex on adverse events. Studies were included in the assessment regardless of study type or setting.The search yielded 19,461 citations; after review, 55 studies were included in the study, involving 28,465 patients treated with adalimumab, certolizumab pegol, infliximab, or vedolizumab. There was no significant association between patient sex and endoscopic efficacy in 41 relevant studies. Increased adverse events were associated with female sex in 7 out of 14 relevant studies.There is no evidence for a sex difference in endoscopically measured response to biological therapies in IBD patients. However, there is an influence of sex on the occurrence of adverse events.The online version of this article (10.1007/s00384-020-03663-2) contains supplementary material, which is available to authorized users. Due to their chronic nature, inflammatory bowel diseases (IBD), consisting of Crohn’s disease (CD) and ulcerative colitis (UC), usually require life-long drug therapies. The treatment paradigm seems to switch, and the current approach has been changed to a more accelerated step-up management of the IBD patient. Currently, a large proportion of IBD patients are treated with biologicals, with studies reporting in the range of 20–25% in Western countries –3, and tHowever, the current evidence on the role of patient sex on the actual response to biological therapies is conflicting. Several studies specifically report on differences in response and adverse events between male and female IBD patients treated with biologicals , 10 wherObjectively measured efficacy, defined as disease activity measured via endoscopy. Examples of this primary outcome include sigmoidoscopy, ileocolonoscopy and capsule endoscopy.Adverse events defined as any adverse event possibly related to biological use. Examples of this secondary outcome are infusion reactions, injection site reactions and hypersensitivity reactions.This study aims to systematically review the literature for studies concerning established biological therapies for patients with inflammatory bowel disease, examining the possible influence of patient sex on:A systematic database search was performed on 08 April 2019, without restrictions on language, publication year or publication status. The search was performed by librarians specialised in database searches. The search was performed in the following databases: Embase (including MEDLINE), MEDLINE OvidSP, Cochrane Central Register of Controlled Trials, Web of Science and PubMed. The detailed digital search strategy is provided in the Supplemental material, Appendix Titles and abstracts identified through the search strategy were assessed by two independent reviewers (ML and EP) for potential eligibility, using pre-defined criteria as described in Study type and methods Participants Interventions Outcomes (including definitions of the primary and secondary outcomes)Data from the eligible studies was extracted using a standardised form by the two primary reviewers. Differences in the extracted data were resolved through consensus or, if necessary, discussion with the third independent reviewer. For each study, the following data was extracted:The risk of bias of included studies was assessed using either the Newcastle-Ottawa Scale (NOS) for cohort studies  or the CP values and/or confidence intervals. If only proportions were reported, the OR was calculated.Results are reported using the summary measure provided by the included studies (e.g. odds ratio (OR), hazard ratio (HR), difference in means) with the respective I-squared test and visual inspection of forest plots. If heterogeneity was present, we attempted to investigate the cause thereof . In the case of high heterogeneity (I2 > 75%), studies were pooled only if the direction of their results was consistent. Subgroup analysis or meta-regression would be performed post hoc, if sufficient studies were included for meta-analysis.For meta-analysis, where applicable, studies were pooled using a random-effects model, regardless of statistical heterogeneity. Heterogeneity was tested using the Chi-squared test, the The literature search performed on 08 April 2019 identified 19,461 citations, of which 11,049 remained after automatic removal of double entries , the risk of bias for the post hoc studies was considered unclear . The three post hoc studies were considered of low-risk of bias, as the original RCTs were of low risk themselves and the safety analyses were pre-specified and used the whole study population . Additionally, female patients reported adverse events as a reason for stopping adalimumab more often than male patients .Two studies were identified that examined patient sex and adverse events during adalimumab use. In a retrospective cohort of CD patients treated with adalimumab for remission induction, Lie et al.  describeIn a large post hoc analysis of 16 RCTs and their open label extensions involving 5345 IBD patients, Colombel et al.  calculatP = 0.001). In a large retrospective study involving 3161 patients treated with infliximab, Ducharme et al. [P < 0.001). Unfortunately, no sub-analysis was performed to assess if this association remains in only IBD patients. Fidder et al. [P < 0.01). Skin lesions were also reported more often in female patients than in male patients . However, no sex difference could be detected for mortality, neoplasia, serious infections, infusion reactions and auto-immune phenomena. The five other studies found no association between patient sex and adverse events during infliximab use.Eight studies described adverse events during infliximab use and patient sex. Three studies found significant associations, with Armuzzi et al.  describie et al.  examinedr et al.  retrospeP = 0.03). This effect was only seen in UC patients, not in CD patients. Furthermore, no association between patient sex and upper respiratory tract infections was found. A general analysis of safety of vedolizumab was performed by Colombel et al. [Two studies examined the possible role of patient sex on the occurrence of adverse events during vedolizumab therapy. In a post hoc analysis of the GEMINI-1, GEMINI-2 and GEMINI open-label extension trials, Feagan et al.  examinedl et al.  using poP = 0.01). Further sub-analyses per drug revealed similar associations, though the association in adalimumab users was not statistically significant, possibly due to low patient numbers. Of note, this study also found that female patients stopped anti-TNF treatment more often than male patients due to adverse drug reactions .In total, two studies were identified that examined the role of patient sex on adverse events during the use of adalimumab or infliximab , 62. OneThe other study by Teriaky et al.  also exaThe objectives of this review were to assess the possible influence of patient sex on biological therapies, on endoscopic outcomes and adverse events in the treatment of inflammatory bowel disease. To our knowledge, this was the first systematic review investigating this research question. With regard to efficacy, none of the studies found an association between patient sex and endoscopically measured efficacy of biological therapies. As for adverse events, half of the included studies found an association between patient sex and various adverse events, with all these studies suggesting that these events occur more frequently in female patients.The intention of this study was to perform a meta-analysis of the included studies; however, several factors precluded synthesis of the data via meta-analysis. Firstly, outcome measures varied amongst studies, with the definitions of adverse events varying from ‘any adverse reaction’ to ‘severe infections’. Secondly, the time-point at which outcomes were measured differed amongst the studies. Thirdly, the study populations were heterogeneous, with some studies examining biological naïve patients and others biological experienced patients or post-operative patients. Fourthly and most importantly, many studies simply reported that patient sex was not associated with the studied outcome, but without providing exact summary measures  or the exact frequencies in which the outcome occurred in male and female patients, respectively. This prevented us from calculating summary measures to perform meta-analysis.Pharmacokinetic studies in IBD patients concerning infliximab , 69 and However, we found no evidence for a sex difference in objectively measured endoscopic disease outcomes. This strongly suggests that biological therapies are effective regardless of patient sex, probably because the underlying inflammatory pathways affected by these therapies are not significantly different between female and male IBD patients. The lack of a sex difference in efficacy of biologicals is also seen in rheumatology patients , 73 and Nevertheless, there have been consistent reports of a sex difference in IBD patients treated with biologicals, with decreased drug survival (i.e. the proportion of patients still using the drug after a set period of time) in female patients , 77. HowThis ambiguity is also present in patients treated with biologicals for dermatologic or rheumatologic conditions. For instance, in psoriasis patients some studies reported more adverse events in female patients , 81 wherThere are several limitations to this study. Concerning the primary outcome of objectively measured efficacy, the included studies varied greatly in their outcome measures. For instance, in CD patients some studies used Simpe Endoscopic Score for Crohn’s Disease (SES-CD) whilst others used Crohn’s Disease Endoscopic Index of Severity (CDEIS), and in UC patients, some studies used the endoscopic Mayo score whilst others used Ulcerative Colitis Endoscopic Index of Severity (UCEIS). Furthermore, even amongst studies using the same outcome measure, the definitions of response and remission could vary. Additionally, there was great variation in the timing of the endoscopic assessment across the included studies. Though this issue was identified during the review, it was decided to include all studies regardless of the heterogeneity of the outcomes. Though a more stringent set of inclusion criteria regarding endoscopic outcomes would have reduced heterogeneity, it was decided to be as inclusive as possible in order to detect a potential signal concerning sex differences. Furthermore, given the lack of meta-analysable results, using more stringent criteria would not have resulted in a different conclusion.The issue of high heterogeneity also occurs in the studies included for the adverse event analysis. Similar to the primary outcome, it was decided to use broad inclusion criteria in order to detect a potential signal concerning sex-differences in the occurrence of biological related adverse events. However, of the seven studies that report a sex difference, in three studies the relation between the analysed adverse events and the drug used is debatable. Firstly, Colombel et al.  find a lIn summary, this systematic review finds no evidence for differences in efficacy of biological therapies in female or male IBD patients, as judged endoscopically. Therefore the sex of the IBD patient need not be directly taken into account when considering starting biologicals or optimisation of biological trough levels. The results concerning adverse events are ambiguous, with half of the studies finding an increased occurrence of adverse events in female patients treated with biological therapies, whereas the other half does not. Extra vigilance and proper counselling for treatment emergent adverse events might be warranted. Further investigations of possible sex differences in the occurrence and severity of adverse events could result in more accurate individualised therapy advice and thus improve the quality of personalised medicine.ESM 1(DOCX 60 kb)


Cannabis has been associated with deficits in memory performance. However, the neural correlates that may underpin impairments remain unclear. We carried out a systematic review of functional magnetic resonance imaging (fMRI) studies investigating brain functional alterations in cannabis users (CU) compared to nonusing controls while performing memory tasks, complemented with focused narrative reviews of relevant preclinical and human studies. Twelve studies employing fMRI were identified finding functional brain activation during memory tasks altered in CU. Memory performance studies showed CU performed worse particularly during verbal memory tasks. Longitudinal studies suggest that cannabis use may have a causal role in memory deficits. Preclinical studies have not provided conclusive evidence of memory deficits following cannabinoid exposure, although they have shown evidence of cannabinoid-induced structural and histological alteration. Memory performance deficits may be related to cannabis use, with lower performance possibly underpinned by altered functional activation. Memory impairments may be associated with the level of cannabis exposure and use of cannabis during developmentally sensitive periods, with possible improvement following cessation of cannabis use. Cannabis is the most-used illicit drug worldwide , with maBrain-structural alterations in cannabis users have been previously attributed to underlie deficits in memory performance. Reduced hippocampal volumes have been observed in cannabis users ,9,10, wiThe cannabinoid 1 (CB1) receptor is the main central cannabinoid receptor through which the leading psychoactive component of cannabis, delta-9-tertahydrocanabinol (THC), exerts its effect. The CB1 receptors are expressed ubiquitously throughout the brain , althougMemory is a multidimensional construct and may be classified based on temporal characteristics into short-term  and long-term memory ; its content  or stage  . DeclaraPrevious work has reviewed both the cognitive ,52,53,54-Original peer-reviewed data-based publication, reported in the English language.-Compared habitual, otherwise healthy cannabis users (>50 occasions of self-reported lifetime cannabis use) with healthy controls (<50 occasions of self-reported lifetime cannabis use).-Used fMRI in conjunction with a memory-based cognitive activation task.A systematic search of previous studies comparing brain functional differences in CU and NU and employing fMRI in conjunction with memory processing tasks as activation paradigms was completed using the PUBMED database following the Cochrane Handbook  and the Studies were excluded if they did not use a cognitive activation paradigm or did not include a memory-based task; did not clearly indicate the extent of cannabis use in the cannabis user group; or involved use less than or equal to 50 times in their lifetime in the cannabis user group; were non-English-language studies.Studies investigating memory performance in humans and animals and brain-function alterations related to memory processing using neuroimaging techniques other than fMRI in humans were identified through a bibliography search of previous systematic and narrative reviews ,58,71,72n = 267) and NU (n = 261) using fMRI were identified in total. All included studies are reported in n = 72), NU (n = 79), while the remaining investigated adults CU (n = 195), NU (n = 182) with a group average age over 20 years. Four papers investigated spatial memory [The initial search for fMRI studies comparing CU to NU while they performed memory-based cognitive activations tasks carried out in October 2015 identified 598 manuscripts. Of those, 10 met our inclusion criteria. Two further studies were identified by our final search on 22/1/2020 ,74, and l memory ,78,79,80l memory ,81,82,83l memory ,84,85,86l memory  and falsl memory  were invl memory  while eil memory ,79,81,83l memory . Three studies investigated spatial memory in adults ,79,80 usAssociative memory in adults was assessed by four studies ,81,82,83Two studies investigated working memory ,85, wherUsing a task used to investigate brain activation associated with false memory, Riba et al.  found CUOnly one study found activation differences in the hippocampus  during tFinally, a study not meeting our entry requirement for cannabis use levels compared 18- to 22-year-old cannabis users, based on their use over the previous 3 months, with those who had not used over that period. Using a visual memory task, no difference in activation was seen in the ROI of the IFG and hippocampus during the encoding condition; however, WBA found CU had decreased activation in the cerebellum (left), insula, basal ganglia, superior frontal gyrus, right precentral gyrus, and bilateral parahippocampal gyri. During the recognition condition of the task, ROI analysis showed CU had significant decreased activation in the hippocampus bilaterally and left IFG, while WBA revealed that CU had decreased activation in the cerebellum , insula, basal ganglia and cingulate, and left posterior parietal cortices . A longiOf the studies in adolescent cannabis users, one used a spatial working memory test and reported decreased activation in frontal and parietal regions in adolescent CU compared to NU . AnotherOnly two studies have employed neuroimaging techniques other than fMRI to investigate neurofunctional differences between CU and NU in the context of memory processing. Battisti et al.  investigSeventeen cross-sectional studies were identified that investigated the effects of cannabis use on memory performance by comparing CU and NU using various cognitive tasks engaging different domains of memory . Twelve Four studies employed a verbal learning task where stimuli were visually presented, finding that CU performed significantly worse at recall of words ,96,97,98Auditory verbal learning tasks were used by six studies, where word lists were read out to the participants. CU had worse recall performance than NU , with hiThree studies used the California verbal learning test ,102,103,Using the Wechsler memory scale, Pope et al. compared a set of heavy-using CU to NU at four time-points of abstinence, finding no significant difference between CU and NU after 25 days of abstinence , replicaNone of the four studies investigating visual memory found a significant difference between CU and NU ,98,101. Two studies investigated auditory verbal learning in adolescent CU compared to NU ,105. SolThe California verbal learning test was used by two studies with one finding some deficits in adolescent CU compared to NU following a 1 month abstinence , while tBoth immediate and general memory performance was tested by Fried et al. in current heavy (average 12.4 (9.8) joints per week), light (<5 joints per week), and former cannabis users (over 3 months abstinence), with all three groups compared to NU separately. Heavy CU performed worse in both immediate and general memory performance, whereas light CU and former CU had no significant difference in performance compared to NU . Two studies investigated working memory in adolescent CU compared to NU ,108, witOne study investigated spatial learning performance following a 1 month abstinence in a group with cannabis use ranging between 60 and 1800 times per lifetime, with no significant difference in performance between CU and NU . We identified six studies that used some form of longitudinal study design to investigate whether memory deficits seen in CU predated the use of the drug or developed following cannabis use. In one of the earliest reports, Fried et al. controlled for differences in cognitive performance prior to initiation of drug use and compared immediate, general, and working memory performance between heavy CU and NU. Heavy CU performed significantly worse in all memory domains compared to NU. Immediate and general memory impairments persisted after controlling for pre-drug-use performance, though working memory performance was no longer significantly impaired after controlling for pre-drug-use performance . In an 8A total of 18 animal studies were identified in our search (listed in Spatial memory, investigated using learning maze-based tasks, was been found to be impaired in rats following chronic exposure to THC or CB1 receptor agonist by five studies ,124,126.Impairments have also been observed in short-term memory in eight studies ,122,123,Of the studies that exposed the animals during adolescence to adulthood, four found that cannabinoids had a negative effect on memory performance in the adolescent group, but not in the adult group in the same study ,119,122.Investigating memory performance after a period of washout is useful to disentangle the residual effects of cannabinoids from any acute effects. Of the four studies that included a memory test done within 24 h of the last drug administration, all reported deficits in the cannabinoid-treated animals ,123,124.Our objective was to carry out a comprehensive review of alterations in brain functioning and performance in the domain of memory associated with persistent cannabis use, by drawing upon evidence from human and relevant preclinical studies. To this end, first, we carried out a systematic review of studies investigating brain-functional alterations in CU compared to NU using fMRI. We also reviewed published studies that have employed neuroimaging techniques other than fMRI to investigate memory-related functional alterations associated with cannabis use. Finally, to situate this understanding within the context of specific subdomains of memory affected, we performed two focused narrative reviews of studies investigating alterations in memory performance associated with persistent cannabis use employing a range of designs: studies in humans using cross-sectional and longitudinal designs to compare CU and NU and preclinical studies comparing cannabinoid exposed animals with nonexposed animals. These results are discussed under separate subsections below.Our systematic review of functional brain activation during memory performance found CU to have altered brain activation, although no consistent pattern emerged either in terms of the direction of alteration in activation or the brain regions affected, although changes appeared to be mostly focused in the frontal and temporal regions. Altered activation in the hippocampus was found in some studies, particularly those employing ROI analysis approaches focusing on the hippocampus, but without a conclusive direction of change. Alteration of hippocampal activation was perhaps less frequently observed than one would have expected considering the central role of the hippocampal region in memory processing. Activation was altered more often during the encoding/learning stage than while recalling information, similar to previous evidence investigating memory performance .Of the three studies reporting hippocampal activation differences between CU and NU during learning, there was no consistent direction of change ,82,83, dThe level of previous cannabis use might have been a potential confounder in studies that have reported no significant differences between CU and NU. Significant differences in activation between CU and NU were observed in studies using similar tasks investigating CU with high levels and prolonged periods of cannabis use ,76,80,87The literature on brain activation differences measured by fMRI between CU and NU during memory tasks still lacks clarity, possibly due to heterogeneity of both the extent of cannabis use as well as the quantity, frequency, and age of onset of cannabis use, which have been found to correlate with alterations seen in cannabis users ,104,131.Functional activation differences between CU and NU were more consistently seen in adult populations of cannabis users than in adolescents, although a previous meta-analysis of all cognitive domains has identified functional difference in CU compared to NU in both adults and adolescents ,63. LackAcute effects of THC confounding previous literature cannot be ruled out in some studies that have investigated participants who were not confirmed to have a negative result for THC on urine drug screening and/or studied participants after only short periods of abstinence from cannabis. Although a sustained period of abstinence may have alleviated some of the group differences in functional activation, abstinence periods did not consistently predict the detection of group differences in functional activation. While this may suggest that certain functional alterations are more robust than others and therefore detectable in CU even following a washout period of the drug, it is very likely that this also reflects the possibility that functional alterations observed cross-sectionally are not just attributable to the effects of drug exposure but an interplay with baseline differences between CU and NU that predate initiation of cannabis use. We reviewed studies of both adults and adolescents, comparing the performance of memory tasks between CU to NU. Verbal memory performance was negatively affected by cannabis use ,105,106,Evidence from some studies suggests that longer duration of cannabis use may have an adverse impact on memory performance ,107 withAbstinence from cannabis use reversed some memory deficits observed, with an earlier meta-analysis showing no significant effect of the drug after a 4-week abstinence on performance . We alsoIt is of interest to note that the effort made during memory tasks has also been found to be negatively associated with frequency of cannabis use ,136. TheWhile some longitudinal studies of memory performance suggest that CU may be associated with a deficit in memory performance following prolonged use, the evidence is not unequivocal, especially when baseline cognitive ability predating initiation of cannabis use is taken into consideration. McKetin conducted a follow-up study finding that CU performed the same irrespective of whether they had continued to use or had ceased use . This maAlthough poorer premorbid memory performance may be partly attributed in some users to their lower cognitive attainment in the domain of memory performance, improvement in performance observed following the cessation of cannabis use suggests that cannabis use may in fact have a direct deleterious effect. Further longitudinal studies are needed to tease apart the effects of cannabis use from other genetic and environmental effects. Other studies have aimed to address the question of the causal nature of the relationship between cannabis use and memory performance and have employed study designs that allowed them to account for genetic and other environmental confounders. A monozygotic twin study by Lyons et al.  investigWe found that animal studies have investigated the effect of not only extracts directly obtained from dried cannabis plants with varying potency in earlier experiments ,120, butThe period of adolescence in rat models is short and so makes it difficult to administer drugs for long enough periods of time when targeting them in the developmental period. However, many studies did attempt to administer drugs for the majority of the adolescent period and saw significant results. The lengthier period of adolescence in humans allows for much longer exposure during neuronal maturation and possibly presents an extended period during which cannabis may have a greater detrimental effect on cognition. Preclinical studies with shorter periods of administration of drugs found fewer deficits in memory, but the period of washout from drug administration seemed to have a major influence on the outcome of studies. The hippocampus is a key region for memory performance , and aniBehavioral tests in animals have not provided conclusive evidence of memory deficits following cannabis use, although structural and histological investigations have shown robust evidence of cannabinoid-induced changes. Collectively, the body of research completed in animals investigating the effects of chronic cannabis use and its possible effects on memory function appears limited in comparison to that investigating the acute effects of cannabinoids. To summarize, cannabis use has been shown in some studies to negatively affect memory-related brain functioning and task performance, particularly verbal memory and encoding in human studies, with preclinical evidence generally consistent with human evidence. Effects have also been observed in recall and working memory tasks, though these findings have been less robust. However, existing evidence regarding the effect of cannabis use on memory function is far from unequivocal, as evident from the heterogeneity in conclusions from the different studies. From the evidence reviewed above, three clear factors emerge that may underlie differences in results seen in studies of memory function following cannabis use. Firstly is abstinence/washout period from last use, which seems to vary a lot in all the animal and human studies reviewed. Longer abstinence periods do appear to be associated with a less pronounced difference between CU and NU participants and cannabis-exposed and -unexposed animals, although not in all cases, particularly following a long exposure. Collectively, results from human studies reviewed here showing recent cannabis use being associated with alteration in memory-related functional activation, which becomes less prominent following periods of abstinence and longitudinal data , suggestSecondly, cannabis use parameters, such as use of higher quantities, longer periods of use, and more regular use, appear to increase the chances of detecting differences in memory-related outcomes. Some, but not all, studies do report linear relationships with time and quantity of cannabis used. Future studies should therefore aim to systematically investigate associations with these parameters of cannabis use and also incorporate other parameters, such as type of cannabis used, that have been shown to be associated with other health outcomes . InteresFinally, the age at which cannabis use starts seems to be another important determinant, with animal literature in particular providing robust evidence for adolescence being a period of higher risk of brain alterations from cannabinoid exposure. From a biological perspective, this is a period of neuronal developmental processes, including brain development and altering binding affinity of CB1 receptors ,65,66,67The detrimental effects of cannabis may be due to CB1-receptor-mediated disruption of hippocampal plasticity, a finding supported by animal histological investigations. Although there is a lack of evidence of significant effects on hippocampal activation from human studies, changes in hippocampal cerebral blood flow and ERPs ,93 as weOne limitation inherent to studies investigating chronic cannabis users, as in any studies investigating recreational drug users and that affects this systematic review as well, is the bias associated with retrospective recall of usage pattern and type of cannabis consumed over time. This is particularly important as the longer-term effects of cannabis use may depend on the specific usage pattern as well as type of cannabis, particularly the ratio of different cannabinoids, which may have often opposing effects on brain function and connectivity ,154,155.To conclude, evidence summarized here suggests that memory performance deficits may be related to cannabis use, with lower performance in memory tasks possibly underpinned by altered functioning of a wide network of brain substrates that may result from changes at the synaptic level. This review did not summarise how functional connectivity may be altered in cannabis users, as was discussed in a previous review , which r


Body iron levels are regulated by hepcidin, a liver-derived peptide that exerts its function by controlling the presence of ferroportin (FPN), the sole cellular iron exporter, on the cell surface. Hepcidin binding leads to FPN internalization and degradation, thereby inhibiting iron release, in particular from iron-absorbing duodenal cells and macrophages involved in iron recycling. Disruption in this regulatory mechanism results in a variety of disorders associated with iron-deficiency or overload. In recent years, increasing evidence has emerged to indicate that, in addition to its role in systemic iron metabolism, FPN may play an important function in local iron control, such that its dysregulation may lead to tissue damage despite unaltered systemic iron homeostasis. In this review, we focus on recent discoveries to discuss the role of FPN-mediated iron export in the microenvironment under both physiological and pathological conditions. C. elegans [Nearly all forms of life require iron for key physiological and developmental processes, such as oxygen transport, energy production and cell proliferation . Only a  elegans . Evidenc elegans  and indi elegans .The control of iron balance is particularly crucial in the context of infection/inflammation, in which pathogens use multiple mechanisms to acquire iron, whereas the host sequesters iron and starves them of this essential metal. The universality of this response is highlighted by the appreciation that this competition for iron occurs also in plants, as iron sequestration is important for plant immune responses .Macrophages, which handle a large proportion of the daily iron turnover, play a key role in the regulation of iron availability in mammals . MacrophSince iron is a double-edged sword, sophisticated mechanisms have evolved to maintain iron balance both at the systemic and cellular levels. Cellular iron homeostasis is regulated through multiple control mechanisms, but the iron regulatory proteins (IRP)-dependent post-translational regulation, which controls the expression of proteins involved in iron uptake , storage (H and L ferritin subunits), release (ferroportin (FPN)) and utilization , is probably the most relevant . Body irHepcidin binding inhibits iron release by triggering FPN internalization and degradation  and occlIn these erythroid precursors, FPN is highly expressed and exports the iron presumably made available by both TfR1-mediated uptake and heme degradation; the latter could be catalyzed by heme oxygenase, which is expressed in erythroblasts , and/or Disruption in these regulatory mechanisms results in a variety of disorders associated with iron deficiency or overload. Notably, iron deficiency anemia, which affects two billion people worldwide, represents the most common nutritional disorder.FPN seems to be localized in a strategic position at the crossroads of these pathways. Over recent years, increasing evidence has emerged to indicate that, in addition to its role in systemic iron metabolism, FPN may play an important function in local iron control, such that its dysregulation may lead to tissue damage despite unaltered systemic iron homeostasis. In this review, we discuss the importance of local iron availability in the microenvironment as an essential factor to maintain tissue homeostasis or repair. Particular attention is paid to iron recycling by macrophages, which is essential for erythropoiesis and may also be relevant for iron redistribution to neighboring cells at the local tissue level .FPN (SLC40A1), a member of the large solute carrier gene family, is a trans-membrane protein expressed ubiquitously, primarily in macrophages and duodenal and hepatic cells, as described above. The finding that mice with global FPN inactivation died during embryonic development confirmed its role as the only (or major) iron exporter (reviewed in ).The structure of FPN and the molecular mechanism of iron export are not entirely clear, but an accepted model indicates that membrane FPN extrudes ferrous iron, as suggested by the facilitation of iron export exerted by ferroxidases, which allow successive ferric iron binding by transferrin . InteresProbably because of its non-redundant role, the synthesis and activity of FPN are subjected to multilevel control . FPN expLess well-characterized transcriptional mechanisms responding to Toll-like receptor (TLR) activation control FPN downmodulation in inflammation, a stratagem preventing pathogen access to iron . An intrAt the post-transcriptional level, FPN synthesis is controlled through the IRP/iron regulatory element (IRE) system . In factHow strong and important are the contributions of each of these pathways to the control of FPN expression? The mechanism based on the interaction between hepcidin and FPN, which leads, for example, to infection-dependent hypoferremia in inflammation, is considered to be the major regulator of iron release, as shown by the relatively small hypoferremia in hepcidin knockout mice . HoweverMutations in FPN lead to disorders of iron metabolism characterized by substantially diverse phenotypes . Gain-ofWith regard to animal experimental models, mice with cell-specific FPN knockout or knockin of hepcidin-unresponsive mutants of FPN have been instrumental for increasing our understanding of the role of FPN in regulating iron levels (see examples below). Indeed, total body inactivation of FPN as well as cell-targeted inactivation of FPN in specific cells resulted in local and/or systemic iron deficiency, sometimes leading to anemia. In addition, the use of cell-specific hepcidin inactivation made possible the characterization of autocrine/paracrine mechanisms controlling iron export based on hepcidin secretion by non-hepatic cells and interaction with FPN on the surface of the same cells.FPN is mainly expressed in the cells involved in systemic iron homeostasis reported above, but it is also expressed ubiquitously and, especially, by tissue resident macrophages. Therefore, it may play an important role in local iron control and its dysregulation can lead to tissue damage despite unaltered systemic iron homeostasis. We provide here some recent and significant examples, which may be representative of the role of FPN in local iron regulation. Tissue specific regulation of iron availability in other organs has been recently covered .Iron control is essential for cardiac function and can be affected by local or systemic defects. Indeed, loss of FPN-mediated intestinal iron absorption may result in iron deficiency and cardiac damage see . On the The analysis of mice with cardiomyocyte-targeted ablation of the FPN coding gene has generated conflicting results: one study showed iron accumulation within cardiomyocytes and consequent heart dysfunction leading to fatal dilated cardiomyopathy despite an unaltered systemic iron level and downmodulation of TfR1-mediated iron uptake, thereby showing that FPN-dependent iron release is necessary to maintain cardiac iron homeostasis . ConversFPN activity in the intestine plays a well-known key role in body iron homeostasis . Given tS. pneumoniae, which require iron for survival and growth [Chronic lung diseases—in particular chronic obstructive pulmonary disease (COPD), which encompasses chronic bronchitis, small airways disease and emphysema, predominantly associated with bacterial infection—are among the leading causes of death worldwide, accounting for about 10 million deaths every year. The pathogenesis of COPD remains poorly understood, but involves inflammatory responses of the lung, most commonly triggered by cigarette smoke. Accordingly, a strong body of evidence implicates a prominent role for alveolar macrophages as drivers of chronic inflammation in COPD. Abnormal iron metabolism has been proposed as a candidate factor involved in COPD susceptibility, with evidence supporting a role for both iron deficiency (and related anemia) and overload. Pulmonary dysfunction has been associated with altered iron balance  and disrd growth .Although the connection between iron dysregulation and lung disease, such as COPD, is undeniable, additional research is needed to elucidate the underlying molecular mechanisms, particularly the role of FPN-dependent iron export.The growing fetus requires a considerable amount of iron, which is supplied by the mother. The syncytiotrophoblast is a polarized cell layer in the placenta which mediates this iron transport. Accordingly, in this tissue, FPN is highly expressed on the basolateral membrane where it exports to the fetal vasculature maternal iron acquired at the apical surface through TfR1. In this context, FPN plays a non-redundant role, as indicated by the demonstration that, when its export activity was preserved in only the placenta, the embryonic lethality observed in mice with global FPN knockout was rescued . The essThe suppression of maternal hepcidin normally occurring during pregnancy is essential for iron transfer to the embryo and prevention of adverse outcomes. Under iron-limited conditions, FPN-dependent iron export to fetal circulation is limited to the amount not needed to satisfy the request of the syncytiotrophoblast, a mechanism largely controlled by maternal hepcidin and hence also influenced by inflammatory conditions which induce hepcidin . This seA recent study showed that hepatocyte-specific expression of NCOA4, the protein that directs ferritin to autophagosomes and lysososomes, is required for erythropoiesis . ObviousThe development of alopecia has been associated with iron deficiency in humans  and was FPN-mediated modulation of iron availability in the tissue microenvironment, independent from systemic regulation, may be relevant in a number of pathophysiological situations. Here, we focus on recent discoveries and report some relevant cases.In the perennial fight between microbial pathogens and host, the competition for iron is crucial and macrophages are the major players in so-called innate nutritional immunity . TherefoFPN is also a major player in the iron-immunity crosstalk; interference with iron acquisition by pathogens is a critical component of the immune response and is a widespread mechanism, present also in plants . SeveralLactobacillus species in the gut microbiota according to intestinal iron content, by inhibiting HIF-2α, impair iron absorption, thereby regulating systemic iron homeostasis [Recently, it has been shown that the crosstalk between host and microbes does not occur only in the context of infectious diseases. In fact, metabolites produced by eostasis .Given the requirement of iron for cell growth , cancer Iron requirements seem particularly evident in cancer stem cells  which, despite constituting a small fraction of tumor cells, exhibit properties—such as unlimited self-renewal, mesenchymal characteristics, drug- and radiation-resistance and the ability to seed metastases—that are important for tumor progression. The alterations of iron homeostasis, including FPN inhibition, that have recently emerged as key factors in cancer growth and progression are present also in CSCs of a number of different tumors . InteresUpregulation of iron import and downregulation of FPN-dependent iron export and iron storage may result in higher iron availability in cancer cells and consequent faster cell growth. However, it should be considered that enough iron must be available in the microenvironment to achieve an efficient uptake, whereas the tumor microenvironment is nutritionally scarce and characterized by limited concentrations of key resources such as oxygen and iron. To cope with these challenging conditions, cancer cells rely on cells in the tumor microenvironment that are essential to tumor growth. Indeed, cancer cells are able to induce the shift of tumor-associated macrophages (TAMs) to a M2-like phenotype, which is characterized by high FPN levels and an “iron donor” phenotype that eventually fosters tumor growth . Not allUsing mice with iron retention in macrophages due to targeted inactivation of FPN, we investigated the role of macrophage iron release in wound healing, a complex process leading to major clinical problems if impaired. Lack of macrophage FPN led to delayed skin wound healing with defective granulation tissue formation and diminished fibroplasia. Iron retention in macrophages had no impact on the inflammatory processes accompanying wound healing, but affected stromal cells proliferation, blood and lymphatic vessels formation, as well as fibrogenesis . In lineThe hepcidin–FPN axis seems to play a role in the modulation of the inflammation and polarization of macrophages present in atherosclerotic plaques, but the effects are different in the various stages of the long process of atherosclerosis progression from early lesions to advanced plaques. Indeed, in early- and mid-stage plaques, pro-inflammatory M1 macrophages are predominant (due to lipid ingestion) and increased iron retention caused by low FPN activity could result in increased ROS production, higher oxidized LDL uptake and decreased cholesterol efflux, ultimately leading to plaque progression . InformaAdvanced complex lesions are characterized by angiogenesis with formation of fragile vessels and hence intraplaque hemorrhage. In this context, macrophages showing an M2 phenotype have high hemoglobin content derived from engulfed erythrocytes but are not iron-overloaded and produce low amounts of ROS because of their high FPN expression, which appears to be directly regulated by liver X receptor LXRα, a nuclear receptor known to activate the transcription of genes involved in cholesterol efflux .. Recently, the availability of a mouse model, obtained by crossing mice lacking ApoE, which spontaneously develop atherosclerosis, with mice with macrophage-specific FPN deficiency, contributed to highlighting the role of FPN-mediated iron export from macrophages. Iron retention in macrophages increased ROS production, inflammation and plaque lipid composition, thereby remarkably accelerating the progression of atherosclerosis. Notably, FPN deficiency in macrophages inhibited the transcription factor LXRα and the expression of its target genes involved in cholesterol efflux, thus promoting foam cell formation and enhancing plaque progression [Iron excess could paradoxically favor the activity of the prolyl hydroxylases and HIF-1α degradation, thus counteracting angiogenesis and leading to plaque stabilization . Recentlgression .In recent years, we have seen tremendous advances in understanding the pathobiology of iron homeostasis, including the role of FPN-mediated iron release. It is increasingly appreciated that these pathologic conditions are related not only to systemic dysregulation, but also impinge on tissue iron balance, mainly dependent on macrophages, which are critical determinants of local iron supply to the microenvironment.How can these recent insights into the pathogenesis of iron-related diseases be translated into clinical practice? For example, regarding iron supplementation, hitting the right dose that would provide iron without inducing hepcidin and hence inhibiting FPN-mediated iron release from reticuloendothelial and duodenal cells is a difficult challenge. For this reason, the major emerging therapies seem to be aimed at the manipulation of the hepcidin–FPN axis, using hepcidin agonists or FPN inhibitors for iron overload disorders characterized by inappropriate/low hepcidin and hepcidin antagonists to favor iron release; as, for example, in anemia of inflammation. The hepcidin/FPN agonist/antagonist therapy approach can be also envisaged for the management of iron trafficking in tissue microenvironment.To date, major efforts have been focused on affecting hepcidin, both directly  and indirectly through the BMP/BMPR pathway (reviewed in ). Howeve