As a leading cause of mortality and morbidity worldwide, cardiovascular disease and its diagnosis, quantification, and stratification remain significant health issues. Increasingly, patients present with cardiovascular disease in the absence of known risk factors, suggesting the presence of yet unrecognized pathological processes and disease predispositions. Fortunately, a host of emerging cardiovascular biomarkers characterizing and quantifying ischaemic heart disease have shown great promise in both laboratory settings and clinical trials. These have demonstrated improved predictive value additional to widely accepted biomarkers as well as providing insight into molecular phenotypes beneath the broad umbrella of cardiovascular disease that may allow for further personalized treatment regimens. However, the process of translation into clinical practice – particularly navigating the legal and commercial landscape – poses a number of challenges. Practical and legal barriers to the biomarker translational pipeline must be further considered to develop strategies to bring novel biomarkers into the clinical sphere and apply these advances at the patient bedside. Here we review the progress of emerging biomarkers in the cardiovascular space, with particular focus on those relevant to the unmet needs in ischaemic heart disease. The burden of cardiovascular disease (CVD) has steadily increased over the past 30 years, contributing to significant morbidity and mortality . Often dIn this section we aim to summarize emerging cardiovascular biomarkers, their discovery, validation and commercialization journey. These have been grouped by their associated pathophysiological processes – atherosclerosis and platelet dysfunction, myocardial ischaemia, fibrosis and remodeling, deranged hemodynamics and contractility, and cardiometabolic disease – visualized in Approximately three quarters of acute coronary events are linked to atherosclerotic plaque rupture . DespiteThe extracellular matrix proteinase, matrix metalloproteinase-9 (MMP-9), is overexpressed in stressed endothelium and is a known contributor to plaque architecture and composition . MMP-9 iIn a cohort of 343 ACS patients requiring ICU admission, MMP-9 levels predicted short-term mortality while both its levels and rate of change during recovery predicted likelihood of a second major cardiac event over a 6 year follow up period . LikewisAdiponectin is a peptide hormone derived from adipose tissue that has protective cardiometabolic effects . This isSerum adiponectin levels are thought to also have a direct limiting effect on atherosclerosis by inhibiting key processes in atheroma formation. This includes improving endothelial cell function and suppressing smooth muscle proliferation and foam cell formation . A case-P-selectin is a cell-surface adhesion molecule expressed on endothelium, platelets, and leukocytes, and has been implicated in plaque development . In partLectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) is physiologically expressed in low amounts on endothelial cells, and sits upstream of several matrix metalloproteinases and pro-inflammatory pathways implicated in atherosclerosis . ReceptoThe prospect of developing an atherosclerotic stability marker or series of markers detectable in peripheral blood may open the door to applying risk stratification or population-based screening estimating risk of plaque rupture or other myocardial event. It remains to be investigated whether these markers used in combination may give additive insight into predictability of short-term or long-term plaque rupture.In addition to its key role in the development of atherosclerosis and vascular dysfunction, inflammation is also seen early in response to myocardial ischaemia. Crucially, these states may be present prior to ACS or within the first minutes from onset, making inflammatory mediators particularly promising cardiovascular biomarkers for detection of acute cardiac events .Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine involved in the early myocardial response to ischaemia . In a moNourin is a potent chemoattractant peptide released by ischaemic myocardium within 5 min of onset . In a coThe established inflammatory “prodrome” preceding acute myocardial events presents an opportunity to detect – and subsequently manage – myocardial ischaemic events in the earliest stages of injury . As thisThe heart's response to ischaemic insult through fibrosis and subsequent remodeling is a principal determinant of it's long-term function . The abiOsteopontin (OPN) is a is a glycoprotein expressed in various cardiac cells that may exist in either an “immobilized” state bound to the extracellular matrix, or as a soluble cytokine mediating inflammatory integrin activation . This abOsteopontin also triggers the secretion of Galectin-3 , a pro-fibrotic agent released from macrophages . In factHigh sensitivity cardiac troponin is one of the most widely used cardiac biomarkers in current clinical practice . Its rolThe essential amino acid phenylalanine has been shown play a role in normal cardiac aging, cellular senescence, and fibrosis. Cell culture studies suggest a causative effect between phenylalanine and heart disease in mouse and human models through promoting aging-related redox and epigenetic changes . A recenCardiac fibrosis is classically an irreversible step in the progression of disease with therapies only indirectly managing hemodynamics and fluid status . HoweverCompromised myocardial contractility and/or relaxation is a hallmark of established cardiac disease . In factCircular RNAs are particularly promising blood biomarkers due to their resistance to degradation compared to other elements of the blood transcriptome . Myocardvia the application of echocardiography and tissue doppler techniques that are impractical to apply at an asymptomatic population screening level  is a complex mediator of angiogenesis with seemingly mixed pro- and anti-angiogenic activity depending on its molecular context , 73. AngIn a large population of 1,315 patients, proteomic analyses identified Ang2 and TSP2 as two particularly promising biomarker candidates across multiple cohorts. In dyspnoeic patients presenting to the emergency department, both were independently useful in distinguishing acute heart failure from alternative causes of dyspnoea after accounting for NT-proBNP level . SecondlGrowth hormone (GH) and insulin-like growth factor 1 (IGF-1) are involved in intracellular calcium handling, myocardial contractile proteins, and systemic vascular function . IGF-1 bTaken together, these markers are promising as a means to assess ventricular function across a range of patient groups. The above trials demonstrate potential in asymptomatic, known heart failure, and post-infarct cohorts. Further exploration of these molecules may also provide mechanistic insights into phenotypes such as ALVD and HFpEF, which are poorly understood .Metabolic syndrome is a key culprit in the modern-day heart disease phenotype . DiabeteLipids are implicated in atherosclerosis both locally in foam cells and the necrotic lipid core, and systemically as mediators of inflammation . In a coMitochondria account for almost a third of the cardiomyocyte by volume – in keeping with their essential role in both cardiac health and disease . LIPCAR Oxidative stress and lipid peroxidation have been implicated in atherogenesis in multiple studies by contributing to endothelial dysfunction and being a strong predictor of CAD events , 88. HowHomoarginine is a nonproteinogenic amino acid that is suspected to have a protective cardiometabolic effect , 93, 94.A more recent study of 2,106 patients reported several of the same correlations, however, when a Mendelian randomization approach was instituted, no significant causal associations of cardiometabolic or vascular protective effects were observed . AlthougThese markers stand to carry increasing importance given the growing recognition of multiple phenotypes within cardiometabolic disease . As suchBiomarkers are applied across the full spectrum of clinical cardiovascular care. In the case of coronary artery disease and ischaemic heart disease, this extends from primary prevention to the diagnosis and risk assessment of acute coronary syndrome, to secondary prevention. Currently, screening in primary care is focused on traditional risk factors such as hypertension and dyslipidaemia, but there is an unmet need for markers that integrate these with the host response. Screening for risk factors and coronary artery disease may help target efforts in patient health education and behavior modification early in disease processes. In the setting of an acute coronary syndrome, some markers are focused on accurate diagnosis , 98. CurThe classical “one size fits all” approach to CVD treatment based on early epidemiological data stands to be replaced by precision medicine, with therapies tailored to the individual patient . The groWhere many major cardiac risk factors including diabetes, hypertension, and dyslipidaemia all have observable outcomes to guide treatment regimens, current standard of practice lacks the ability to practically measure case-by-case potency of antiplatelet therapy , 102. ThAnother panel proposes 16 biomarkers to guide the choice of antihypertensive pharmacotherapy in the context of heart failure. Several of these have already been shown to have prognostic value such as BNP, NT-proBNP, ST2, Gal-3 or cardiac troponins , 106. AnFurthermore, there may be scope for this biomarker panel to guide the choice of pharmacotherapy. An analysis of 499 patients from the TIME-CHF study showed the relationship between different therapies and biomarkers . One of Cardiac resynchronisation therapy (CRT) – when successful – may improve quality of life, mortality, hospitalization, and NYHA class in select HF patients, and even partially reverses detrimental cardiac remodeling , 111. A Lipid-lowering statin therapy is widely used in cardiovascular secondary prevention, making for one of the most widely prescribed drugs in the world . A lipidCommercialisation pathways play a critical role in allowing for the rigorous translation of biomarkers or other medical innovations to the clinic . UltimatThe ability to patent biomarkers and consequently enforce said patents is a complex issue that has undergone significant recent regulatory changes, and vary between countries and regulatory bodies. Notably in the United States, three key decisions in Mayo v Prometheus, AMP v Myriad Genetics and Alice Corp. v CLS Bank International placed significant restrictions on what biomarker claims could be patented .The Mayo decision of 2012 highlighted how natural processes (in this case being the measurement of a metabolite to titrate drug dose) cannot be patented The Myriad decision of 2013 demonstrated that naturally occurring products (in this case the detection of DNA fragments) cannot be patented .The Alice decision of 2014 demonstrated that abstract ideas  cannot be patented .Thus, current and future claims will need to pass three tests to be patent eligible: first, that it differs from nature or natural processes; second, that it consists of inventive concepts ; and third, that it differs significantly from routine clinical practice . DespiteIn addition to patents, there are alternative pathways for a novel marker being implemented in the clinic that include licensing of “know how” and platforms or tools to rigorously measure the marker and communicate results to physicians and patients. As detailed in Despite restrictions on patenting biological processes, there remain a number of pathways to commercially incentivise emerging biomarkers. Patents may still be taken out on inventions that utilize biomarkers to produce clinically relevant results, including assays. An example of this is the cardiac troponin – no single patent is held on its detection but a number of “high sensitivity” assays have been patented by separate groups over the years, resulting in increasing sensitivity through a competitive commercial environment while still providing financial incentive . This haSimilarly, novel platforms for detection and quantification emphasizing rapid delivery of results are also grounds for patenting. Aptasensors are a form of biosensors using highly specific oligonucleotides or short peptides to bind to a target analyte and produce a signal through a transducer that can then be quantified . AptasenTranslation of biomarkers from the laboratory into the clinical sphere brings with it a number of technical challenges. The most obvious of these is validation of laboratory findings in relevant clinical cohorts in a reproducible manner, but there are several other hurdles to consider . DetectiBroadly speaking, a given biomarker should provide additional predictive value over established tests, typically requiring improved sensitivity and/or specificity to be demonstrated . HoweverFinally, there is the hurdle of approval for clinical use by governing regulatory bodies. Indeed, of the biomarkers discussed in this paper only galectin-3, sST2, and hsCRP have recognized cardiovascular indications by the FDA despite the promising human clinical data presented above , 137. ThThere remains a considerable unmet need for an improved armament of blood-based biomarkers in early detection and prevention of a variety of common cardiovascular disease states. Against this, hurdles to the development and commercialization of cardiovascular biomarkers remain. The markers presented above carry promising prognostic and predictive value that suggests applicability in guiding therapy, however longitudinal data demonstrating a reflection in improved patient outcomes over time remains limited and should be an area of future attention. Improved molecular phenotyping with more advanced blood-based biosignatures will likely permit enhanced stratification of patients in subgroups where distinct biology may be shared, and subsequently drive personalized treatment regimens. Further, the growing understanding of these disease phenotypes provided by emerging biomarkers may implicitly lead to the development of therapies specifically targeting these subgroups. This has the potential to assist in accelerating translation of biologically relevant therapies – such as novel small molecules and targeted RNA therapies – in groups enriched with relevant signaling abnormalities. Ongoing collaboration between academic and clinical leaders with industry and regulatory bodies are required to overcome hurdles in biomarker translation. Looking forward, exciting opportunities abound in this area that will enable the practice of cardiology to better embrace the precision medicine strategies of the future.SS and JG drafted the initial version under the supervision of GF, with all other authors contributing additional sections and editing. All authors were significantly involved in the writing and editing of this 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Reactive hyperemia is a well-established technique for the non-invasive evaluation of the peripheral microcirculatory function, measured as the magnitude of limb re-perfusion after a brief period of ischemia. Despite widespread adoption by researchers and clinicians alike, many uncertainties remain surrounding interpretation, compounded by patient-specific confounding factors (such as blood pressure or the metabolic rate of the ischemic limb). Mathematical modeling can accelerate our understanding of the physiology underlying the reactive hyperemia response and guide in the estimation of quantities which are difficult to measure experimentally. In this work, we aim to provide a comprehensive guide for mathematical modeling techniques that can be used for describing the key phenomena involved in the reactive hyperemia response, alongside their limitations and advantages. The reported methodologies can be used for investigating specific reactive hyperemia aspects alone, or can be combined into a computational framework to be used in (pre-)clinical settings. At the local level, the microcirculation continuously regulates the levels of blood flow and pressure across its network to satisfy metabolic demands, to redistribute hydraulic loads and to promote inflammatory processes. To this end, a sophisticated hierarchical control system  operates by regulating the diameter of the microvessels, causing vaso-dilation or vaso-constriction, which in turn modulates their hydrodynamic resistance. When a significant increase in tissue metabolic demand occurs, a large fraction of the microcirculation needs to be recruited for coordinating the vaso-dilation. To achieve this, the vaso-dilation arising from the lower microcirculation can ‘ascend’ into feed arteries by means of an electrical signal conducted along the endothelial cells (ECs), hyper-polarizing vascular smooth muscle cells (SMCs) causing relaxation.121 Indeed, it is within the microcirculation that the earliest signs of several cardiovascular diseases manifest themselves. For example, the Firefighters and Their Endothelium (FATE) study8 showed that microvascular health represents a powerful independent predictor of cardiovascular events in primary prevention. Thus, assessment of microvascular function may not only have utility for providing novel insights into the pathophysiology of the patient, but also presents an important opportunity for early disease detection and risk stratification.7Since microcirculation represents the ‘mesoscale’ functionally bridging the systemic circulation to perfused tissues, microvascular dysfunction (MVD) ultimately compromises oxygen delivery to the end organ(s).108 Invasive approaches can indeed selectively partition macro- from microvascular function but are not suitable nor feasible in all individuals. While non-invasive CT, PET and MRI address many of these concerns, exposing participants to ionizing radiation, confined spaces, and/or strong magnetic fields also limits widespread adoption. As such, there has been considerable focus on the development of safe and cost-effective alternative methods for measuring microvascular reactivity.123 Accordingly, much attention has been placed on the peripheral vasculature, as it is easily accessible and reasonably reflects vascular function in other end-organs of interest.90 Although not yet established in clinical practice, methods such as reactive hyperemia provide prognostically-significant indicators, capable of differentiating clinical phenotypes.107 In its simplest form, reactive hyperemia represents the magnitude of limb reperfusion following a brief period of ischemia induced by arterial occlusion , magnetic resonance imaging (MRI) and positron emission tomography (PET).105 and tissue perfusion by near-infrared diffuse correlation spectroscopy (NIRS-DCS)13; each of which with its own strengths and weaknesses.107 As detailed in this review article, the determinants of reactive hyperemia are multi-factorial and difficult to quantify. Thus, the best measurement approach is one that accounts for the greatest number of such complexities.Fundamentally, the approach measures microvascular vasoreactivity to metabolic substances produced in response to tissue ischemia. Here we consider reactive hyperemia to be limited only to the initial increase in flow that immediately follows the restoration of flow. Indeed, the blood flow response after this initial period is affected by regulatory mechanisms that fall outside of pure ‘microvascular function’. Multiple methods exist for evaluating reactive hyperemia, including limb distension by venous occlusion plethysmography, blood velocity/flow via Doppler ultrasound of an upstream conduit vessel, kinetic changes in tissue oxygenation by near-infrared spectroscopy (NIRS),Computer modeling can be used as a complementary tool for hypothesis testing, making predictions and quantifying the dynamics underlying vascular regulation that cannot be directly observed during in vivo experimentation. Here, we report on various modeling techniques used to describe reactive hyperemia dynamics across the circulation, and describe the effects of various assumptions on hierarchical blood flow control system. The review concludes with an integration of knowledge, relating the theoretical modeling with existing data collected by our group and others , identifying key opportunities for future discovery.Occlusion of a conduit blood vessel, like the brachial artery, has a direct negative impact on the resulting pressure and flow distribution downstream of the occlusion. The reduction in pressure quickly propagates from the occlusion site down to the level of the exchange vessels, ultimately impairing oxygen delivery. The ensuing tissue ischemia results in microvascular vasodilation , such that when the occlusion is reversed, the ensuing blood flow is markedly elevated (i.e. reactive hyperemia).53 or simplified one-dimensional (1D) blood flow modeling approaches which consider field variations only along the main flow  direction.88 The latter methodology is less accurate , but has many competitive computational advantages and can be easily adopted for vessel networks.5 This makes 1D blood flow modeling the best option for describing the fluid mechanics in reactive hyperemia, which involves propagating phenomena along the vasculature with time scale of many seconds. Furthermore, through 1D blood flow modeling it is also possible to perform wave intensity analysis which allows the quantification of pressure waveforms travelling forward to the microcirculation and backward to the heart.88 In arteries (and arterioles) the blood behaviour can be generally approximated as homogeneous and Newtonian since the size of the red blood cells carried by the plasma is considerably smaller (10 times) than the vessel diameter. Furthermore, flow is also generally considered incompressible and laminar, with a Poiseuille velocity profile. In 1D blood flow modeling, each compliant vessel can be treated as axisymmetric, with blood velocity (u), pressure (P) and flow described as continuous variables along its axial direction z. These quantities are averaged over the cross-sectional area (A) and their variation along the radial direction is considered negligible. The Navier-Stokes equations for 1D blood flow in compliant vessels can be expressed in terms of cross-sectional area and velocity averaged over the cross-section:t is time, 26 or cross-sectional area and flow rate.112 The mechanics underlying the vascular wall deformation appears to be complex, mainly due to vessel visco-elastic properties and the capacity to produce active tone for diameter regulation. To describe the interaction between blood and vessel wall, different approaches can be used.113 In the simplest case, the fluid pressure is related to the cross-section of the vessel via a linear function with respect to the luminal diameter  and the geometric (diameter and length) and structural (stiffness) wall properties can be made vessel specific and can reflect different physiological and pathological conditions such as ageing or hypertension.88 The blood flow variables described by system  fluid-structure interaction modelsensional D blood fensional D blood fentclass1pt{minima111 provides a detailed characterization of the flow through microcirculatory networks. In these microvessels the Reynolds numbers are < 1, and therefore the blood flow can be described with a good level of accuracy as incompressible Stokes fluid, for which the convective component is neglected. Blood flow can still be described by  appears to be associated with the tendency of RBCs to migrate away from the vessel wall.111 The resulting blood viscosity can be evaluated as a product between the plasma viscosity and the relative apparent viscosity  where the nucleus is treated as bulky deformable solid. These multidimensional models allow the direct assessment of the physics underlying the interaction between RBCs and microvessel wall but they require efficient parallel algorithms95 for being scalable to realistic and physiologically relevant problems.Opposite to the continuum approach, blood flow across microvessels can also be described via motion of multiple discrete particles119 0D models may be employed alone for describing the full cardiovascular system or parts of it and can be represented through electrical analogy. Lumped models can also be used in combination with higher-fidelity counterparts to represent those vascular territories where a low level of space details is required.Lumped or zero-dimensional (0D) modeling represents an approach in which the system’s quantities are not (directly) space-dependent but depend only on time. Their governing equations can be directly derived from formulations of high-fidelity/space-dependent models  and the space-dependent characteristics are encapsulated through a set of parameters which are generally identified using experimental data.92 split the vasculature into different flow compartments, each of them characterized by specific parameters representing vascular features such as flow resistance and compliance. In this case, the full haemodynamic profiles across the vasculature can be obtained by simply computing a set of (time-dependent) ordinary differential equations. This approach involves very low computational costs and allows an easy integration and/or comparison with quantities averaged over space obtained from some experimental imaging techniques (such as doppler ultrasound or NIRS-DCS). Therefore, due to its simplicity, 0D modeling should be considered as first-step for modeling blood flow variables during reactive hyperemia. On the other hand, lumped modeling alone cannot adequately capture the wave-propagation phenomena underlying the reactive hyperemia response and therefore does not provide a suitable platform for a full mechanistic understanding of the different space-dependent interactions between vascular territories. Nevertheless we re-iterate that, for various questions related to reactive hyperemia, lumped modeling should be considered for the description of the whole system’s dynamics, with space-dependent models introduced as ‘sub-models’ for further refinement if necessary.Compartmental lumped models115 or generated by using branching (either deterministic or stochastic) algorithms.74 From CT or MRI scans it is possible to obtain, after segmentation, networks of large blood vessels17 but diameters smaller than 500 115 To facilitate the integration of experimental datasets in network models, methods were developed for classifying microvessels without requiring any flow information115 or estimating flow rate with unknown boundary conditions55 The microvascular arrangement and consequent levels of blood perfusion strongly depend on local morphological and functional properties of the tissue such as 131 Skeletal muscles are perfused by bundles of parallel capillaries which transport blood from a common terminal arteriole to the same post-capillary venule. Mendelson et al.76 used intravital video microscopy to show that capillary modules may present substantial structural differences in terms of topology, as well as functional heterogeneities in RBCs haemodynamics within the same module and across modules. This study proposed a new paradigm for capillary flow organization which is pivotal for explaining the complex flow regulation occurring during skeletal muscle contraction. Capillary modules are indeed interconnected to form continuous columns, defined as capillary fascicle, that align with the structure of the muscle fascicle.76 Blood flow through the arterial-venous vasculature (arteries 74The topology of the fluid network can either be reconstructed from images4). For computing blood flow in large arteries during reactive hyperemia, the inlet boundary condition could be imposed either at the heart level or at a  brachial artery site (as long as the inlet is not beyond the occlusion site). However, the latter case is practically less feasible than the former as it requires the prescription of both flow and pressure (which are difficult to measure simultaneously) for not omitting the potential contribution of backwards reflections. The left ventricle  can therefore be used as the inflow prescription point,113 with its complex pumping function simplified using a lumped model, which is able to capture the essential features of inflow data.To compute haemodynamic variables along a portion of the vasculature, it is necessary to define the boundary conditions at the network extremities to represent the remaining part of the cardiovascular system that is not explicitly modelled in space  tree approach,91 its derived versions,94 and the porous-media based approach.36 While these models encapsulate more details of the downstream network, they come with higher computational costs.To close the fluid network, it is always necessary to define appropriate outflow conditions, which represent the haemodynamics characterizing the downstream systems. Venous hydrodynamic conditions  may play a significant role in the experimental settings,73Tissue ischemia generates hyperpolarization of the smallest microvessels (capillaries mainly) which is then conducted upstream through EC gap junctions to relax the arteriolar vasculature and favour a more consistent/systemic increase in blood flow. In the time period between tissue ischemia generation and the full recovery of the system, intrinsic pressure and flow control mechanisms simultaneously work together to restore homeostasis; the entirety of which is extremely complex and difficult to quantify.107 reports an exhaustive description of the cellular pathways involved from the stimulus generation to vessel dilation. Accordingly, the stimulus originating the hyperpolarization is yet to be defined but most likely involves a combination of 38 Recent findings75 on the capillary EC function showed that membrane hyperpolarization may trigger a complex cascade of 133Tissue metabolism involves the production and consumption of different substances, some of which have been traditionally associated with hypoxia  was simulated by combining the EC 35 are therefore instrumental for deciphering the emergent cell dynamics features. The capabilities of space-homogeneous models can be further extended by adding transport equations for describing diffusion of species127 or models representing cytosolic micro-domains80 for assessing the interaction between sub-cellular components. Space-homogenous models allow an easy extension to cluster of cells for computing the resulting intra-cellular signaling. On the other hand, this would be hard to achieve in case a multi-dimensional approach for cell dynamics was used. Therefore, cellular space-homogeneous models represent the most trivial option as bottom level of a multi-scale vessel framework. Furthermore, et al.60 However, a recent theoretical study104 suggested that, with respect to other OIn the computer model, the originating stimulus can be represented either as a single variable in a set of kinetic equations describing oxygen/tissue signaling or simply as perturbation of the concentration of species in the extracellular space. In the reference model by Moshkforoush 11 According to Welsh et al., the conduction of the signal is enabled by a defined pattern of charge movement along the vascular wall which is the result of the interplay between tissue structure, gap junction resistivity and ion channel activity.125 Despite the vessel axis being the main direction of propagation, the hyperpolarizing signal is also transmitted into surrounding SMCs through myoendothelial junctions to promote vascular relaxation.122 Endothelial 14 Experiments in rat cremaster arterioles indicate that vasoactive conduction is favoured by circulation of autocrine and paracrine mediators ATP and K44Ascending vasodilation allows to export the local capillary hyperpolarization to a widespread fraction of the microvasculature. The endothelium serves as the predominant cellular pathway for signal conduction as the hyperpolarization is transmitted from cell to cell along the vessel wall through gap junctions.et al.,42 in which vascular intercellular gap junctions in the skeletal muscle were represented by ohmic resistors. For investigating the signal transduction involved in conducted vaso-reactivity, Kapela et al. integrated their previous modeling efforts114 into a multicellular model of a rat mesenteric arteriole which comprises a detailed description of ECs-SMCs units, coupled via non-selective gap junctions.70 This framework was used in a subsequent study71 for estimating from experimental measurements the electrical resistance associated to cell membrane and gap junctions. More recently, the same group presented an experimentally-validated computational framework for reproducing the propagation of the hyperpolarizing signal across a cerebral microvascular network.81 This provides some key information regarding how the activity of different ion channels can impact on the ascending signal and its re-generation. However, this framework neither quantifies the dynamic changes in microvascular compliance nor indicates whether the metabolic demand is satisfied but it represents a solid modeling base to start from. Alternatively, Arciero et al.9 developed a multi-compartmental framework based on a previous autoregulation wall mechanics model to represent the adjustments in diameter across a microvascular network induced by vaso-conducted metabolic response. In this work the stimulation considered was the hypoxic-induced ATP release by RBCs and the hemodynamic model was coupled with Krogh-type models for representing the mass exchange with tissue.Early efforts in modeling electrical communication across vascular branching include the works by Diep 19 and studies on brain function63 capillaries are able to control local blood through pericytes contraction. In the study by Horn et al.,65 a feed artery occlusion caused a heterogeneous flow distribution in capillary beds with the presence of capillary no-reflow, and this appeared as result of the complex interaction between myogenic and metabolic mechanisms occurring at the arteriolar level. In simulated capillary modules, RBCs flow appeared to be independent from the network resistance.77 This indicates that RBCs flow across capillaries is mainly regulated at control sites located at-pre and post-capillary levels. Overall, the role of single capillaries on skeletal muscle local flow regulation still needs to be fully elucidated but its extent seems minor with respect to the upstream microcirculation.97 Experimental evidence indicated that also venules and veins are endowed with a contractile apparatus, and therefore they may play a role in the pressure re-distribution occurring during and after the arterial occlusion.120 However, their high blood volume capacity suggests a minor role of these vessels especially in the first part of haemodynamic response after occlusion release.The conducted changes in membrane potential spread from capillaries to arterioles. Indeed, in rabbit skeletal muscle87 A change in the wall membrane potential promotes the increase in vessel diameter in potentially two ways. First, as direct effect, hyperpolarization of the SMC causes a decrease in intracellular et al.38 showed that, in the forearm, there is no combined role of NO and PGs in peak RH. Therefore, EC 79 but these seem to be present only above a certain blood pressure level  into one modeling framework. Jin et al.,67 extended this work by including a realistic blood flow network model. This model closely approximates experimental recordings made at the conduit artery level but its representation of the downstream microvascular dynamics remains limited. Some studies110 have also assessed the impact of microvascular diameter changes on blood rheology and tissue perfusion across different vasculatures. It is however important to note that, despite such models allowing quantification of flow characteristics upon imposed variations in microvessel resistance, there is no direct link with a realistic coordinated regulatory response.To date, there is a limited number of models able to describe the coordinated haemodynamic response to arterial occlusion through a multi-scale vascular system. The work by Yamazaki and Kamiyama132Due to their intrinsic simplicity, compartmental lumped models represent the standard approach for evaluating the blood flow dynamics across different levels of the microvasculature, without directly describing propagation phenomena. For each compartment, vascular changes due to regulation can be represented by varying (in time) its lumped parameters  in calf muscles of human volunteers. Through all these modeling approaches it is generally possible to evaluate quantities that can be directly associated with in vivo experimental measurements carried out in human patients. However, this type of approach requires the condensation of most regulatory mechanisms within few model parameters, which might not have a clear physical meaning and that in most of the times need to be estimated by fitting against experimental data. At the same time, some of these compacted model parameters can be used for a straightforward discrimination of different types of cardiovascular profiles. As such, given the complexity of reactive hyperemia, this class of models is likely most appropriate for capturing the interaction between vascular compartments and their contribution to the regulated haemodynamic response.de Mul 48There is a pressing need to enhance our understanding on microvascular function and its regulation. Advances in this direction will ultimately lead to the development of new diagnostic, progress monitoring and therapeutical approaches for diseases characterized by (micro)vascular dysfunction. Through this work, we sought to provide a guide for modeling reactive hyperemia, which represents a standard method for assessing microvascular reactivity. Based on the current knowledge on reactive hyperemia, we reported a series of models that can be used for describing physiological phenomena that occurs at different space and time scales, spanning from cellular ionic currents to systemic circulation flow dynamics. Several issues hinder the accurate modeling of reactive hyperemia. First and foremost, from an experimental point of view, isolating single vascular control mechanisms and vascular compartment contribution to the resulting haemodynamics is technically very challenging if not impossible. On the other hand, it is possible to measure the combined response at locations across different experimental conditions, in order to best inform the computer model. The use of multiple simultaneous recordings of blood flow via Doppler ultrasound as well as RBCs flux via NIRS-DCS may also be helpful in the identification of model parameters and quantification of their uncertainties.There is need to define, test and use suitable modeling approaches that, despite the complexity of the system analysed, are capable of capturing the relevant interactions between vascular compartments. Furthermore, there is need for computational models that can efficiently deal with different scales in space and time as this aspect is essential for the successful translation of the computer model into clinical practice. Connections between components of the hierarchical multi-scale models play a crucial role. Further investigation and validation is needed before modeling may inform clinical decision making. To this end, high-fidelity models that leverage large existing datasets augmented by machine learning/data-driven approaches may be needed.


Renal transplantation is the gold-standard treatment for adolescents and young adults with end-stage renal disease. Despite enjoying excellent short-term outcomes, they suffer the worst rates of premature transplant function loss. Health behaviors: such as lack of adherence to immunosuppressive medications, are felt to be the major contributory factor. Understanding the educational needs of young renal transplant recipients allows healthcare practitioners to better support patients in managing their chronic disease. The aim of this scoping review was to understand what is known about their educational needs. A scoping review methodology was followed. Following an online search, study titles, and abstracts were screened for eligibility, followed by full-text assessment and data extraction. Data were qualitatively analyzed using thematic analysis. A total of 29 studies were included in the scoping review. In young people who struggled with self-management, three themes were identified (1) the Needs of the disrupted youth, (2) the Needs of the disorganized youth (3) the Needs of the distressed youth. There was a paucity of research to identify the protective factors that enable young recipients to successfully manage their health. This review outlines current knowledge of the patient education needs of young transplant recipients. It also highlights remaining research gaps that will need to be addressed with future research. Children on hemodialysis have a reported 55-fold increased mortality risk compared to the healthy population of a similar age . CardiovFor young people, the benefits of transplantation are multiple and diverse and go beyond the excellent survival benefits. The overall accrual of disease burden is significantly less than when on dialysis, and a functioning transplant graft facilitates normal growth and development . Young aDespite the overwhelming benefits of transplantation, it is also associated with challenges. Many young transplant recipients (up to 30%) show symptoms in keeping with post-traumatic stress disorder (PTSD) after transplantation . PatientAdolescents and young adults enjoy better short-term outcomes following renal transplantation than any other age group . DespiteAlbeit complex and multifactorial, the reason for the high rates of transplant graft loss in this group is often attributed to health behaviors and lack of adherence to prescribed therapies . AdolescGiven the high association between health behaviors and patient education in young people with chronic diseases , it is cA scoping review is defined as “a form of knowledge synthesis that addresses an exploratory research question aimed at mapping key concepts, types of evidence, and gaps in research related to a defined area or field by systematically searching, selecting, and synthesizing existing knowledge” .This study was completed adhering to the methodological recommendations and steps as laid out by Colquhoun et al., 2014, Levac et al., 2010, and Arksey, 2005 ,24,25. TAll primary research related to adolescent (defined as aged 10–18) or young adult (defined as aged 18–24) renal transplant recipients’ educational needs were eligible for inclusion. Professional reports, quality improvement projects, and editorials were not included. Studies where other solid organ recipients were included and data pertaining to renal transplant recipients only could not be separated were excluded. From the research question, a search strategy was developed with the assistance of a medical librarian. Six online databases were searched for relevant articles-OVID-Medline, EMBASE, Web of Science, Scopus, CINAHL, and Psychinfo from inception until 17 December 2022. A targeted grey literature search was also conducted for further articles for inclusion. An example search strategy can be viewed in the Initially, titles and abstracts of selected articles were reviewed for appropriateness for full text review against the inclusion of material by two members of the research team. The full-text review was then conducted independently by two researchers. Disagreements for full-text inclusion/exclusion were solved by consensus or arbitration by a third party. Data were then extracted from each included article using a pre-designed data extraction tool. A completed data extraction tool can be viewed in the Study characteristics: author, year of publication, country, design, sample size, clinical setting, number studiedPopulation characteristics: adolescent vs. young adult, transplant functionStudy design: methods used to investigate educational needsKey findingsThe following information was extracted from each selected study;Both qualitative and quantitative studies were identified, so a mixed methods approach was taken for data synthesis. The approach outlined by the Joanna Briggs Institute for mixed-method data analysis in synthesis reviews was utilized . A conveThe results of the thematic analysis were discussed in a consultative exercise with young transplant recipients and healthcare professionals involved in transplant education to enrichen and validate the analysis. This step aided in refined and bringing together individual codes to form the themes derived in the review and bring the patient perspective to our findings.A total of 2954 records were identified through database searching, and an additional 13 records were identified through grey literature searching. Following duplicate removal and abstract screening, the full text of 394 articles was reviewed. A PRISMA 2020 flow diagram [The needs of the disrupted youthThe needs of the disorganized youthThe needs of the distressed youthThree themes of an investigation by researchers emerged from the studies following the thematic coding of the data:Some young transplant recipients appear to face substantial delays in their education, and their levels of qualifications are lower than their peers . The disStudies also investigated how disruption to their youth impacts young transplant recipients meeting major psychosocial milestones . Young aResearchers suggest that young people who are supported to reach their full potential  seem to enjoy additional protection from anxiety and depression and even have better clinical outcomes, such as improved transplant function .The selected studies focusing on the importance of young transplant recipients developing organizational skills, engaging with their health, and having self-motivation as they hypothesized that this would lead to better transplant and overall health outcomes.The largest reported barrier to developing self-reliance skills in recipients was a lack of knowledge and understanding of their condition and medications. Studies described how recipients feel disengaged with their own health and often are disempowered due to a lack of health knowledge . Those wMany young adult transplant recipients lack coping mechanisms or strategies to help them deal with the extra organizational burden of being a transplant patient . ResearcThis transition from dependent child to independent adult often leaves recipients feeling unprepared, uninformed, and unable to become fully responsible for their health—increasing their risk of complications, especially around clinic attendance, blood sampling, and medication compliance .Studies showed that young transplant recipients craved “normality” and wished to be viewed as similar to their peers . Some yoStudies consistently demonstrated recipients reporting symptoms or diagnostic signs of anxiety, depression, irrational anger, and even post-traumatic stress disorder . PsycholThis review demonstrates the wide range of educational needs of young transplant recipients studied in the literature. Areas such as health literacy, self-management, and organizational skills may appear obvious and have been documented in studies of young people with other chronic physical illnesses . DespiteHowever, educational needs go beyond the recipient’s understanding of their condition and how to take medications. The findings of this review would suggest that young recipients need additional support to meet educational/professional/developmental goals that are disrupted compared to their peers. Young people with other physical health conditions, such as diabetes mellitus , childhoThe review raises questions about how young people are prepared psychologically for transplant. Poor coping skills, disappointment from high expectations of life post-transplantation, and psychological toll all appear to be areas of educational need for young recipients. Given the associative links between mental health outcomes and overall transplant function ,52,53,54Interestingly very few studies investigated protective factors or what allowed some young transplant recipients to be successful in managing the burdens associated with transplantation. From the search strategy, there were no papers that fully explored demographic, social, or psychological factors that were associated with good outcomes . There also appeared to be few studies that explored the lived experiences of young transplant recipients who managed their condition well without complications.(1)There is a paucity of research on how certain young people are successful and cope/adapt following transplantation which may help in developing tools to help other patients.(2)Educational needs of racial, ethnic, and religious minority groups and those from immigrant or socially deprived backgrounds need further exploration as these may be different from the general population.(3)An in-depth qualitative analysis of young transplant recipients, which fully explores the contextual factors such as healthcare setting, background, and resources, may help further our understanding of their experiences and requirements.(4)Future studies that are designed and carried out with adolescent or young adult transplant recipients as equal partners may improve the quality and validity of the results.This review highlights potential areas of research to better understand the educational needs of adolescent and young adult renal transplant recipients:A scoping review is intended to map out and organize the current literature and does not assess the quality of the studies identified. Hence, the review is limited in its ability to confirm the validity of each study’s claim of young recipients’ experiences. However, given the wide-ranging and complex factors that affect educational needs, a positivist review methodology i.e., searching for the ‘right’ answer with a systematic qualitative review, would likely have also been limited in its conclusions. Truthfully, it will be hard for any review to generalize the experiences of adolescent and young adult transplant recipients from varying cultural and social backgrounds and healthcare settings around the globe. Therefore, the authors hope this review, inkeeping with scoping review methodology, will offer an appropriate understanding of the current literature and identify potential research gaps.Support systems and health services need to be designed by clinicians to aid young people, and physicians should have knowledge of other professional services and members of the multidisciplinary team that can assist. Transplant physicians need to be particularly aware of the increased risk of anxiety, depression, and PTSD in these patients and be able to appropriately refer them to other services. Ultimately it is hoped that this review will aid in either targeting research or changing healthcare provision for young transplant recipients to improve their care. It should also highlight the importance of this issue and encourage more service providers to reach out and seek young recipients’ opinions and voices. The findings of this review may be of interest to patient advocacy groups.This scoping review has outlined the literature that addresses the educational needs of younger transplant recipients and highlights the gaps in our knowledge. Hence, for clinical teams to develop best practices in the management of young renal transplant recipients, further research is required. The transplant community needs to prioritize good quality research that is patient-centred and patient-led to better inform practice. In-depth qualitative studies that seek to fully explore the lived experiences of young transplant recipients could provide better insights into their unique educational needs. This, in-turn, could help inform future intervention trials and hopefully improve outcomes for young recipients.


Lung epithelial organoids for the hazard assessment of inhaled nanomaterials offer a promising improvement to in vitro culture systems used so far. Organoids grow in three-dimensional (3D) spheres and can be derived from either induced pluripotent stem cells (iPSC) or primary lung tissue stem cells from either human or mouse. In this perspective we will highlight advantages and disadvantages of traditional culture systems frequently used for testing nanomaterials and compare them to lung epithelial organoids. We also discuss the differences between tissue and iPSC-derived organoids and give an outlook in which direction the whole field could possibly go with these versatile tools. Inhalation is by far the most important route of exposure for airborne pollutants and particles. Pulmonary particle exposure comprises airborne pathogens, including viruses and bacteria, but also ambient particulate matter, such as combustion-derived particles and even engineered nanomaterials (NM); the latter mainly at occupational settings during production, processing or decomposition. Depending on their aerodynamic diameter, airborne particles bigger than a few micrometers are deposited along the surface covered with mucus of the conducting airways by impaction, where they are rapidly removed via mucociliary clearance. Inhaled nanoparticles (NP) smaller than 100 nm in diameter deposit mainly by diffusion in the whole lung, but are especially efficient in the most distal and fragile parts of the lung, the alveoli . The priOnce reaching the alveolar surface, particles can lead to serious health consequences such as attenuated lung development for children exposed to combustion-derived traffic emissions ,8, cardiTo achieve a smarter and more ethical approach to NM testing, the Adverse Outcome Pathway (AOP) framework has been established, which incorporates mechanistic knowledge generated from in vivo experiments to connect measured toxicological endpoints with a pathological consequence by a sequence of molecular initiating events (MIEs), consecutive key events (KEs) and the final adverse outcome . Several AOPs have been identified and shown to have strong correlation across published in vivo datasets . To get In the following section, we will (1) portrait the difficulties of current in vitro models especially for specified AOP based testing, (2) introduce different lung organoid cultures as an alternative method and (3) give an outlook on these NAMs in the field of research.Numerous studies display adverse effects of NM on the lung or lung cells, including cell proliferation, oxidative stress ,17, DNA To overcome these disadvantages for inhaled particles, cells can be cultured on an Air–Liquid Interface (ALI). By placing the cells or tissue on a porous membrane and feeding them just from the basal side, the apical side is open for an inhalation like airborne exposure, thereby a comparable experimental set up to in vivo conditions arises. Hence, in vitro exposure at the ALI with airborne NMs is not only the more realistic approach, but also the one allowing defined cell delivered dose estimations compared to exposure under submerged conditions. ALI inhalation models have the potential for a more precise reproduction of the processes during exposure, as they can mimic the fragile respiratory epithelial region comparable to structural in vivo terms . EspeciaEven with the most desired advanced models, it is noted that the results generated by inhalation of nanoparticles in vivo cannot be fully and properly represented in vitro. Previous studies have shown that the use of immortalized cell lines does not represent the in vivo situation completely, so does not provide fully comparable results to those obtained in vivo. This relates to the fact that immortalized cell lines often lose polarity and lack key morphology features, which may biologically distinguish respective cells in the context of tissue. Furthermore, as the immortalized cells do not have a natural proliferation cycle due to mutation or manipulation, they have evaded normal cellular senescence and instead can keep undergoing division, which could lead to functional alterations and genetic drifts ,34. In gA promising approach to overcome disadvantages of currently widely used immortalized cell lines and to compare results created in vivo with in vitro data is the use of three-dimensional (3D) cell cultures, the so-called lung organoids. Organoids are defined as three-dimensional, mostly spherical shaped constructs cultured in vitro in an extracellular matrix. They self-organize from single stem cells into multicellular structures and mimic the in vivo organ, in this case the bronchiolar or alveolar region of the lung . An overOne method to grow lung organoids is to isolate primary epithelial cells out of lung tissue. This is possible with murine lungs as well as human tissue, although the availability of human lung tissue is limited. Basal cells act as progenitor cells in the tracheal and bronchial region of the lungs . When isAn alternative to primary lung epithelial cells for generating lung organoids is the use of directed differentiation of induced pluripotent stem cells (iPSCs). Since the discovery of human iPSCs , they arThe experimental set-ups of in vitro lung models are based on biochemical differentiation of hiPSCs into lung lineages. Organoids derived from stem cells (ESC or hiPSC) are able to differentiate and self-organize through lineage bonding comparable to processes taking place during development in vivo .hiPSCs differentiated into lung progenitors can be used for deriving airway organoids. They contain SCGB1A1+ secretory cells, multiciliated cells expressing FOXJ1 and basal cells, amongst others . In modiGreat advantages of 3D lung organoid cultures compared to conventional cell lines are the comparable cellular identity and functionality to the in vivo situation, and the potential to differentiate into several epithelial cell types. This enables us to perform disease modeling, developmental and regeneration studies, identify roles of the distinct cell types regarding cellular communication in defined settings and create a representative model of airway and/or alveolar lung compartments. When comparing architecture and functional readouts of lung tissue, a 3D cell culture system creates much better and even more realistic conditions than a cell monolayer culture system ,58. A feAnother approach to take advantage of the stem cell character that cells keep in organoid culture is to dissociate the cultured 3D organoids into single cells again. When cultured in Transwell inserts, organoid derived epithelial cells can form an intact epithelial barrier .In this setting, exposure to NMs using the ALICE system, where particles are nebulized and a defined dose is distributed equally upon the cells, is feasible. The combination of using cells with functions and properties as in vivo, and the inhalation-like exposure to particles with the ALICE system makes this culture method interesting. In summary, the combination of organoid culture and subsequent ALI exposure to balance the limitations of each individual model will be a useful approach to assess NM hazards.Lung organoid technology has developed quickly in the last years and became a useful tool for modeling perpetuating lung diseases and hazards affecting the lung . With reThus, the choice of cells used should be thoroughly considered, especially concerning their respective properties, such as forming lamellar bodies, producing surfactant or retaining stem cell character. It becomes clear that there is not one overall cell line for a general experimental setup, for instance with regard to NM inhalation, where particle–cell interactions in a realistic environment are of particular importance. Therefore, using advanced target cells that are able to create a liquid lining layer would improve the comparability of in vitro studies to in vivo findings and lead to extended outcomes .One important step in the future is to increase the use of stem cell derived murine or, even better, human cells that adequately reflect the disease pattern for monitoring and understanding the underlying cell–cell interactions after NM exposure. For instance, the use of immortalized cell lines within an in vitro experiment has shown to be not comparable to a clinical picture. Isolated human primary cells can only be passaged for a short period of time and are therefore also not sufficiently suitable for a complex experimental set-up with necessary replicates. At this point, an adapted experimental setup with hiPSCs would be a desirable and new promising approach. Due to their close resemblance to the primary cells, despite their durability and the possibility to be passaged over a long period, hiPSC derived organoids should be the prospective choice for human in vitro experiments.In addition, an adequate murine in vitro 3D culture system has several advantages. It is important to create setups reflecting and confirming the findings observed in previous in vivo studies. This enables us to elucidate cell–cell interactions and events happening on cellular, protein and gene levels, while reducing the number of animals used in similar in vivo testing according to the 3R principles. Based on the AOP framework regarding NM toxicity, lung organoid culture could be a helpful NAM to obtain results representing in vivo conditions more accurately. With the emergence of new analytical techniques, profiling cellular responses at the single cell level, we realize that a tissue such as the lung consists of over 50 different cell types . Yet theIn summary, we illustrate that already established experimental setups with new and adapted cells will lead to potentially improved or even new results and findings. Lung organoids include these particular cells, enabling us to perform hazard assessments for NMs within suitable models.


Dear Editor,Since ages, the word ‘testing’ is equated or irreversibly used for assessment purposes and mostly confined to measuring the amount of knowledge gained by the students in the stipulated period of curriculum or assessment of whether students perform up to a satisfactory level. However, recent literature  , 2 sugIn an interesting study involving post-graduate residents, it was found that the cohort who were tested repeatedly were found to have greater retention of learned content even six months after the teaching encounter compared to those who studied repeatedly  . Test-eAdesope OO et al.  when and how should test-enhanced learning be used in instructional models? b) What question formats could be used for that purpose? c) How to align it with educational objectives? Our intention is to highlight the fact that frequent retrieval practice is equally important as how students are taught in the classroom. Practising retrieval at regular spacing enables long term retention and helps to transfer the knowledge across different contexts. Thus, we suggest the educators to incorporate the principles of test-enhanced learning in all phases of the iterative process of learning. In conditions where high stakes examination leads to cramming and creates an illusion of successful learning, test-enhanced learning, if utilized in an effective manner, could act as an effective pedagogical catalyst in complex educational systems. None Declared.


Iatrogenic arteriovenous fistulas (AVFs) and pseudoaneurysms (PSAs) are rare complications that may develop years after vascular access, and high-volume flow through these AVFs have been hypothesized to contribute to chronic heart failure. Formation of an AVF or PSA following Impella placement has rarely been described in the literature. Here, we describe a patient who had percutaneous placement of an Impella ventricular assist device through his right groin three years prior, now presenting with worsening heart failure and symptoms of volume overload. He was discovered to have a new, high-flow common femoral artery to femoral vein AVF with an associated PSA. The AVF and associated PSA were resected and repaired. This case study highlights a rare access-site complication from percutaneous Impella placement associated with worsening heart failure, strategies for preventing this complication during peripheral access, and the need to consider this differential in such a patient with a history of peripheral access who has an unexplained worsening of heart failure. Blood vessel trauma through peripheral access may result in a rare formation of an arteriovenous fistula (AVF) –3. High-The patient is a 51-year-old male admitted to the hospital with shortness of breath and clinical signs of volume overload from acute exacerbation of chronic heart failure with reduced ejection fraction at around 25-30% as determined via echocardiogram. His past medical history is notable for nonischemic cardiomyopathy (cardiogenic shock and biventricular systolic dysfunction) requiring placement of an Impella ventricular assist device through his right groin three years before presentation, paroxysmal atrial fibrillation on anticoagulation, right lower extremity deep vein thrombosis, chronic kidney disease, an implantable cardioverter defibrillator (ICD), and alcoholic cirrhosis. Careful physical exam revealed a palpable thrill on the right groin distal to the inguinal ligament, which suggested a vascular malformation. This area was the same location where peripheral access was obtained for Impella placement. Information regarding access site management after Impella placement is unavailable.Duplex ultrasound was performed for imaging . Of noteNext, surgery was undertaken to resect the AVF with PSA. Swan-Ganz catheter found pulmonary artery pressures to be elevated. Surgical exposure identified a common femoral artery to femoral vein AVF with PSA, shown in Proximal control of the femoral artery at the ligament level and control of the vein at the ligament level were established. The AVF and PSA were mobilized along with obtaining distal artery and vein control . The venWe describe an arteriovenous fistula and associated pseudoaneurysm in a patient with worsening heart failure who had percutaneous placement of an Impella ventricular assist device three years prior to presentation of volume overload symptoms. Iatrogenic AVFs and PSAs may be rare complications years after peripheral vascular access, and prior reports have documented delayed formation of AVFs between femoral, subclavian, and radial vessels due to percutaneous vascular access –3. FurthFormation of an AVF with PSA following Impella placement has rarely been described in the literature. One of the few papers that discuss this briefly mentioned formation of an AVF as a possible access site complication following Impella-supported percutaneous coronary interventions is . However7 Optimally, the puncture site should be 1-2 cm below the inguinal ligament and above the femoral bifurcation [9 Although routine ultrasound follow-up was not performed after Impella placement in our patient, this may be a cost-saving and effective preventative measure to monitor for the complication of AVF formation.Preventative measures can be taken during peripheral vascular access to avoid development of AVF formation. At the inguinal ligament, the common femoral vein is medial to the common femoral artery. However, the vein travels distally more laterally and posterior as it reaches the edge of the femoral triangle, and a distal access site is more likely to injure either or both the artery and vein to predispose to fistula or pseudoaneurysm formation . To avoiurcation . This coOriginally admitted for worsening heart failure symptoms without clear explanation, the patient was only discovered to have an arteriovenous fistula and pseudoaneurysm after closer history detailing Impella placement three years prior and a detailed physical exam revealing a palpable thrill on the right groin in the area of the access site. This highlights an important consideration of the medical history and physical exam in the workup of patients presenting with unexplained symptoms of volume overload who may have an AVF and/or PSA. Medical history should include discussion of prior vascular access and a simple physical exam that searches for any palpable thrills. Positive results can then be followed up with imaging and possible surgical resection as exemplified in this patient.In summary, this 51-year-old gentleman presented with volume overload symptoms secondary to heart failure, found to have a femoral AVF and pseudoaneurysm likely due to percutaneous insertion of an Impella ventricular assist device through the right common femoral artery. This is a rare case of an access site complication after Impella placement that has rarely been documented. We describe the link between this high-flow AVF with PSA and exacerbation of heart failure and strategies for preventing this complication as well as the importance of the history and physical exam for diagnosis.


Cryptococcus neoformans is a fungus that may cause several CNS manifestations, especially in immunocompromised patients. Cryptococcal meningitis is the most common type of involvement. Mass-effect lesions are uncommon: they are described as cryptococcomas and their prevalence is even lower among immunocompetent patients. The aim here was to report an extremely rare case of cryptococcoma causing a mass effect and mimicking a brain tumor in an immunocompetent patient. The literature on CNS cryptococcal infections was reviewed with emphasis on cryptococcomas. Clinical, surgical and radiological data on a female patient with this rare presentation of cryptococcoma mimicking a brain tumor are described. Central nervous system (CNS) infectious diseases have high prevalence in developing countries and their proper diagnosis and treatment are very important for public health planning.  A 54-year-old female patient presented to the emergency department with a rapid-onset progressive history of confusion and completely dependency for basic activities. Neuroimaging showed a left occipital lesion and neurosurgical treatment was proposed. From histopathological evaluation, a diagnosis of cryptococcoma was established. She received clinical support with antifungals, but despite optimal clinical treatment, her condition evolved to death. Cryptococcal infections have several forms of presentation and, in immunocompetent patients, their manifestation may be even more different. Cryptococcoma is an extremely rare presentation in which proper surgical and clinical treatment should be instituted as quickly as possible, but even so, there is a high mortality rate. When this organism passes through the blood-brain barrier, it generally means that the host defenses are compromised. This may be due to human immunodeficiency virus (HIV) infection or to chronic conditions such as renal and vascular diseases.The lungs are the primary sites of infection. From there, Cryptococcus neoformans infection may be manifested as neurocryptococcoma, which is a granulomatous CNS lesion that may cause a mass effect.,In rarer cases, Here, we describe a rare case of a female patient who did not have any condition that reduced her immunity. The only relevant occurrence in her medical history was the presence of controlled arterial hypertension. She presented to the emergency department with a complaint of confusion that evolved very quickly to completely dependence for daily activities. Brain magnetic resonance imaging (MRI) showed mass-effect lesions in the left occipital lobe. She underwent neurosurgical intervention and meningitis treatment. Initially after surgery, she showed some neurological improvement. However, despite optimal treatment, her condition evolved to death.A 54-year-old female patient presented to the neurosurgical emergency department, brought by her family, who described a history of rapid and progressive mental confusion , leading to complete dependence for basic daily activities such as baths and eating. The only significant condition in her medical history was hypertension. One relevant social factor was that she had grown up on a farm and had had direct contact with several bird species including pigeons.General and neurological physical examinations revealed that the patient was normotensive but in a poor general condition, was only able to obey simple orders, was restricted to bed , seemed not to have any motor deficits and did not have any cranial nerves alterations or meningeal signs. It was difficult to perform a complete neurological examination because of her general condition. Based on her history and physical examination, she was categorized as having a score of 50 on the Karnofsky performance scale (KPS).An MRI scan  performeCryptococcus neoformans were observed in the samples, along with increased protein levels). Before the puncture, a brain CT scan was performed to rule out any signs of intracranial hypertension or any alteration that might make it impossible to perform this procedure. All punctures revealed increased intracranial pressure (ICP) that progressively worsened , but she did not present hydrocephalus.After the operation, the patient showed some neurological improvement and, after some days, a lumbar puncture was performed to evaluate the presence of meningitis. The presence of cerebrospinal fluid (CSF) infection was confirmed  and fluconazole was administered, in association with clinical support, because she presented renal failure due to use of many drugs and required hemodialysis. However, despite optimal clinical treatment in the intensive care unit (ICU), comprising antifungal drugs, broad-spectrum antibiotics against pneumonia, electrolyte replacement, hemodialysis and respiratory physiotherapy, the patient’s condition evolved to death due to various complications, including the initial disease, the toxicity of the various medications and bronchoaspiration leading to sepsis.,,,Cryptococcosis is the most common fungal disease of the central nervous systemTransmission occurs through inhalation of substances that are present in mammal and bird feces (pigeon feces have been described most frequently). Thus, the lungs constitute the entrance for the disease and pulmonary impairment is usually the first manifestation.,Central nervous system cryptococcal infections usually manifest as meningitis, meningoencephalitis, encephalitis or ventriculitis.The World Health Organization (WHO) recommends two weeks of induction treatment with amphotericin B deoxycholate (AmBd) and flucytosine or fluconazole, followed by eight weeks of consolidation treatment with oral fluconazole.Cryptococcus can inhibit the migration and function of leukocytes and promote survival and localized replication of the pathogen, thus facilitating chronic granulomatous inflammation and cryptococcoma formation.,,,,,,,,,,,Nevertheless, in rare cases, this chronic granulomatous process can lead to formation of a mass (cryptococcoma) that has a tumoral appearance.,,Surgical excision and debulking are indicated as adjunct therapies for lesions that are greater than or equal to 3 cm.Intraparenchymal cryptococcomas usually present with low signal intensity on T1-weighted MRI and high intensity on T2-weighted images. Solitary cryptococcomas are very rare lesions.The patient of this paper was extensively investigated but did not have any immunosuppressive condition or pulmonary impairment. The only data that might have helped us to think of the diagnosis of cryptococcoma was the patient’s previous contact with pigeons . Because the patient had a neurological dysfunction that developed quickly, which made us think initially that the cause was an aggressive brain tumor, it was decided to perform surgical removal. The histopathological analysis  confirmeDiagnosing the tumoral form of cryptococcosis (cryptococcoma) in immunocompetent patients is a challenge even in endemic regions. Primary and secondary brain tumors are usually the first hypotheses in these cases. Proper investigation through anamnesis and imaging can lead to consideration of this diagnosis before the histopathological analysis has been conducted. A neurosurgical approach should be considered in cases of this type of mass lesion, to reduce the compressive factor and thus the intracranial pressure. Despite optimal treatment, this is a condition with high mortality.


Plasmodium spp.) remains one of the leading causes of morbidity and mortality, especially in tropical regions of the world. Despite the availability of malaria control tools such as integrated vector management and effective therapeutics, these measures have been continuously undermined by the emergence of vector resistance to insecticides or parasite resistance to frontline antimalarial drugs. Whilst the recent pilot implementation of the RTS,S malaria vaccine is indeed a remarkable feat, highly effective vaccines against malaria remain elusive. The barriers to effective vaccines result from the complexity of both the malaria parasite lifecycle and the parasite as an organism itself with consequent major gaps in our understanding of their biology. Historically and due to the practical and ethical difficulties of working with human malaria infections, research into malaria parasite biology has been extensively facilitated by animal models. Animals have been used to study disease pathogenesis, host immune responses and their (dys)regulation and further disease processes such as transmission. Moreover, animal models remain at the forefront of pre-clinical evaluations of antimalarial drugs  and vaccines. In this review, we discuss commonly used animal models of malaria, the parasite species used and their advantages and limitations which hinder their extrapolation to actual human disease. We also place into this context the most recent developments such as organoid technologies and humanized mice.Infection by malaria parasites ( Malaria caused by P. vivax is also highly prevalent in the WHO South-East Asia and America regions  cycle. During this stage, merozoites transit through ring stages to metabolically active trophozoites which massively consumes host haemoglobin and nutrients producing microscopic distinct brown pigments of haemozoin. In mid–late trophozoite stages, the parasite starts replicating its DNA (S-phase) and divides its nucleus to enter the schizont stage. Once mature, schizonts contain, typically, a species-specific 10–30 merozoites, which upon rupture of the infected cell re-invade new red blood cells to initiate another ABS cycle. This results in a cyclic increase in the percentage of infected red blood cells (the ‘parasitaemia’). Schizonts and late trophozoites (of some species) are not usually seen in peripheral circulation as they sequester in the microvasculature  human disease while helping in decoding the critical aspects of parasite biology, immunology, and pathogenesis for development of novel interventions. Consequently, animal models have been historically invaluable to the study of malaria. These have ranged from birds, bats, non-human primates (NHPs), rodents and more recently humanized mice and complex three-dimensional ex vivo organoids. In this review, we explore different Plasmodium spp. which have been used to query human malaria disease in different animal models. With a special focus on NHPs and rodent species, we review the history of human infecting Plasmodium that are able to infect rodents and NHPs. We also outline the history of rodent-specific Plasmodium in the wider context of disease similarity with human malaria. More importantly, the review discusses the aspects of parasite biology and malaria disease processes that these animal models are helping to unravel. We also explore some recent advancements in controlled human infections with human P. falciparum and the consequent implications on future use of different animal models in malaria research.Despite frequent exposure to malaria parasites, ABS long lasting, stage transcending immunity is not known to occur in malaria infections. This has made malaria vaccine development especially challenging despite the recent rollout of the moderately effective but short-term RTS,S vaccine  cause disease in humans. Despite its discovery as the main causative agent of malaria in humans in 1880 by the French Army physician Charles Laveran, P. falciparum did not inherit its widely used name  until its certification by the International Commission on Zoological Nomenclature over 70 years later in 1954 following years of debate on the naming system   that infect splenectomized or non-splenectomized monkeys from the American tropics have been reported and are archived by the US Centre for Disease Control  are becoming attractive and suitable models as a potential substitute to NHPs. They are generated by transplantation with either primary human hepatocytes or red blood cells under immunodeficiency conditions to prevent xenorejection. Because various organs and or tissues can be transplanted into mice, this is becoming a more convenient approach to modelling several aspects of malaria parasite biology as well as disease pathogenesis. However, humanized mice still lack sufficient tissue or organ penetration of the engrafted cells due to the hostile mouse microenvironment for foreign human cells to survive and proliferate. Moreover, the use of immunocompromised mice limits the study of certain aspects of host–pathogen interactions such as host immune responses. Nevertheless, as the development of these humanized mice continue to improve, their utility and full potential will hopefully be realized, reviewed in detail by Kaushansky et al.  that were originally isolated on various occasions in Central African thicket rats have been at the centre of these studies. These parasite species share a highly conserved chromosomal gene synteny with the human infecting P. falciparum   have been extensively used to study and model human disease P. ovale . Neverth humans) . Even th et al., .Fig. 2.P. cynomolgi and P. vivax hypnozoites (dormant liver stages) were first discovered in NHPs, specifically in rhesus macaques for the former   have been successfully adapted providing, in the absence of long-term in vitro culture, the only means to generate sufficient amounts of parasite material for downstream analyses  has been characterized and preserved by the CDC ever since  are known to sequester in tissues and organs just like human P. falciparum. P. coatneyi infected red blood cells also exhibit rosetting characteristics as is commonly seen in P. falciparum  in macaques  that often results in a rapid increase in parasitaemia and death of the host in the absence of treatment. Even though this parasite can infect humans, in what has been some of the best-described cases of zoonotic malaria , avian malaria parasites were the experimental spp. of choice for studying malaria parasite biology as they were discovered at almost the same time as P. falciparum  which is endemic to all parts of the world except Antarctica  that undergoes antigenic variation  model, mice develop similar neurological features to human CM which among others include paralysis, coma and death  revealed that parasite-infected red blood cells occluded brain micro-vessels, a feature which was not observed in similar mouse infections with mild disease  severe and lethal forms of the disease. Severe malaria is often characterized by a multi-organ system involvement presenting as anaemia, metabolic acidosis or cerebral malaria (CM)  which seemingly mediate parasite adhesion properties and antigenic variations  protein were shown to provide immune protection to mice by blocking sporozoite-mediated invasion of hepatocytes  result in robust humoral as well as CD4 T-cells immune responses capable of blocking follow-up challenges with wild-type sporozoites  further alludes to the complications of using such models to broadly inform on the liver stage biology of all HIPs. Thus far, P. cynomolgi remains the only robust NHP malaria parasite that is extensively used to study P. vivax liver stage biology due to their close relatedness  , IgG antibody responses, T helper 1 cells and proinflammatory cytokines (Interleukin 12 and interferon λ) have been shown to control the onset of acute infection and progression to peak parasitaemia , malaria animal models have also been instrumental in understanding host immune responses and the biology of erythrocytic blood stages. Particularly, RMPs together with longitudinal studies of experimental human infection challenges  have been remarkably useful in dissecting the liver and blood-stage biology of malaria parasites. This is in addition to host immune responses to these stages which are the basis of current malaria vaccine developments through either the attenuated liver and ABSs or their subunits   and P. chabaudi  immunogen conferred protective immunity to the host in human-compatible vaccine dose formulations  were also used (for the first time) to demonstrate parasite antigenic plasticity to monoclonal antibody inhibition in some of the early malaria target-based immunization strategies. Immune sera to P. knowlesi PK140 resulted in rapid evolution and replacement of antibody binding epitopes with mutant forms  and toxicities. Such profiles fine tune the discovery pipeline by selecting antimalarial agents with better potency and safety landscapes in a hit to lead optimization strategies. Animals have also been used to decipher the genetic basis of antimalarial drug resistance through in vivo drug selection and more recently to validate some in vivo phenotypes of orthologous drug resistance markers identified in P. falciparum.In antimalarial drug discovery programmes, animal models of malaria are widely used to evaluate in vitro whole cell phenotypic screens of Plasmodium parasites within host red cells  have become an option recently . Scaffolds with better profiles in the above assays are further refined to achieve better antimalarial potency or improve their ADME and toxicity safety scores. New scaffolds which pass the established benchmarks are then tested for in vivo efficacy and safety, usually in a small animal model. These in vivo proofs of concept studies often utilize RMPs, particularly P. berghei and P. yoelii  and P. falciparum could be due to reticulocyte preferences for the former which provide rich metabolic energy sources with the potential to cushion the killing effect of antimalarial agents  and in vivo (P. berghei) sensitivities for some inhibitors and or whether such discrepancies are due to intrinsic differences between the parasite species or due to the pharmacodynamic/pharmacokinetic properties of the drug in the animal model. Since proof of concept in an animal model is usually required before any drug discovery programme is progressed, RMPs have, indeed, been at the front of these evaluations as most antimalarial drug candidates in clinical development at the moment  were all shown to cure mice infected with P. berghei , RMPs are uniquely suited for such endeavours due to the ease of handling rodents and the ability to reproduce the entire in vivo life cycle under lab conditions   carrying pyrimethamine resistance alleles are currently a widely used drug selection marker for transfection experiments in both P. berghei and P. falciparum  line. This line was obtained by a further selection of the AS-Pyr line with a single four-day high dose exposure with the SP drug combination to obtain parasite progenies that were strongly resistant to the drug combination. However, quantitative trait loci and genetic analysis of the AS (50S/P) line revealed that dhfr mutations were the major determinant of the SP drug resistance phenotype as no additional dhps mutations were identified   was obtained and cloned  was obtained   resulted in the parasite line that was initially resistant to up to four consecutive doses of mefloquine at 30 mg kg−1 but eventually lost some degree of resistance by only surviving four consecutive doses of the drug at 15 mg kg−1 (designated AS-15MF)  was obtained  yielded an ART-resistant line (AS-ART) that survived three to five consecutive doses (up to 300 mg kg−1) of the drug  appear to have fixed independently in the AS-ATN (V2697F), AS-30CQ and AS-ART line (V2728F)  that seemingly was responsible for the chloroquine resistance phenotype in the AS-3CQ line and another mutation (T719N) in a predicted unknown transporter (PCHAS_0313700) which appears to have led to the emergence of the chloroquine resistance phenotype in the AS-15CQ line  in P. falciparum clinical isolates that have presented with potential ART resistance phenotypes  are frontline antimalarial drugs in ART-based combination therapies, their deployment of which has played significant roles in alleviating the global malaria disease burden WHO, . Howeveroroquine . The oriRosario, . Selecti) Padua, . From thet al., P. berghei, P. yoelii and P. chabaudi  is readily achieved with the acquisition of mutations in the cytochrome bc1 complex both under laboratory conditions and in clinical field settings  have been implicated as a mechanism of resistance  as the principal determinant of resistance involved detailed genomic, biochemical and allele exchange experiments to identify a 13-exon gene within a 36 kb chromosomal fragment that strongly associated with chloroquine resistance phenotypes in resistant parasites  that could be a possible determinant of this drug resistance phenotype  which, crucially, displayed high-level resistance in both the P. berghei in vitro short-term assay as well as in vivo  are easily amenable to genetic manipulation as they demonstrate higher transfection efficiency as compared to P. falciparum gene dosage or if the background expression is sufficient to overshadow any resulting phenotype from the introduced alleles. However, with the development of recent highly precise genome editing technologies such as CRISPR-Cas9, instead of introducing P. falciparum antimalarial drug-resistant candidate alleles in RMPs as transgenes, orthologous polymorphisms can be introduced with precision in attempts to characterize phenotypes based on assumed gene function conservation. Such CRISPR approaches have recently been used in P. berghei in vivo to validate UBP-1 and Kelch13 ART resistance mutations which were first identified in P. chabaudi and P. falciparum, respectively , there appears to be a reasonable degree of similarity in the functional domains  all resulted in ART or chloroquine resistance phenotypes just like their P. falciparum and P. chabaudi equivalents which were reversible when some of these mutations were reversed or repaired  possessed fitness defects which are easily assessed and quantified in RMPs under physiologically relevant in vivo conditions. In SEA, Kelch13 mediated ART resistance is primarily associated with the C580Y mutation  mutation and the other important Kelch13 mutation, R539T (R543T) could not be introduced in this RMP in several attempts despite a high degree of conservation of the predicted Kelch13 structures and mutation locale between the two parasites  in P. berghei have facilitated the identification of critical drug and vaccine targetable pathways throughout the Plasmodium life cycle, a wealth of information which is difficult to generate for HIP (Gomes et al., et al., et al., et al., et al., et al., et al., et al., et al., P. falciparum in vivo data ranging from host immune responses to parasite infection, drug and vaccine evaluation as well as the basic biology of life cycle progression. However, these first-generation humanized mice suffer from poor permeation of engrafted cells while recipient mice retain some core features of immune cell compartments. These can be limiting to the study of certain aspects of parasite biology and the development of the next generation humanized mice with humanization in several tissue and organ compartments will hopefully refine and enrich host–pathogen interaction research outputs from these models (Minkah et al., et al., P. falciparum strains typically used in the controlled infections as well as the fact that most field infections are known to comprise of multiple strains is also a limiting factor and a scientific hurdle controlled human infection approaches must overcome. The recent development of liver-derived organoids has also provided an additional ex vivo tool set for probing the biology of Plasmodium liver stages in host cultures that closely mimic in vivo conditions, reviewed by Mellin and Boddey (P. falciparum and P. vivax liver stages opening up exciting avenues in downstream liver stage biology, drug discovery and host–parasite interactions. Nevertheless, for meaningful and exhaustive outputs, harmonized and complimentary approaches to malaria research, which utilize in vitro approaches with HIPs and traditional animal models in parallel with the emergent controlled human infections is the ongoing approach that will hopefully arm the next decades of the fight against malaria.Laboratory malaria research has always relied on animal models which have served as d Boddey . Liver o