In recent years, a growing interest has arisen regarding the possible relationship between adverse pregnancy outcomes (APOs) and inadequate maternal hemodynamic adaptations to the pregnancy. A possible association between “placental syndromes,” such as preeclampsia (PE) and fetal growth restriction (FGR), and subsequent maternal cardiovascular diseases (CVD) later in life has been reported. The two subtypes of FGR show different pathogenetic and clinical features. Defective placentation, due to a poor trophoblastic invasion of the maternal spiral arteries, is believed to play a central role in the pathogenesis of early-onset PE and FGR. Since placental functioning is dependent on the maternal cardiovascular system, a pre-existent or subsequent cardiovascular impairment may play a key role in the pathogenesis of early-onset FGR. Late FGR does not seem to be determined by a primary abnormal placentation in the first trimester. The pathological pathway of late-onset FGR may be due to a primary maternal cardiovascular maladaptation: CV system shows a flat profile and remains similar to those of non-pregnant women. Since the second trimester, when the placenta is already developed and increases its functional request, a hypovolemic state could lead to placental hypoperfusion and to an altered maturation of the placental villous tree and therefore to an altered fetal growth. Thus, this review focalizes on the possible relationship between maternal cardiac function and placentation in the development of both early and late-onset FGR. A better understanding of maternal hemodynamics in pregnancies complicated by FGR could bring various benefits in clinical practice, improving screening and therapeutic tools. Fetal growth restriction (FGR) takes place when the fetus does not achieve its biological growth potential . EtiologThe relationship between fetal growth and placental development has been extensively studied since the last century . ThroughHowever, signs of abnormal spiral artery remodeling may be observed in normal pregnancies, as well as normal spiral artery remodeling may be seen in cases affected by FGR .Therefore, a non-placentocentric point of view should be considered to fully understand the pathophysiology of FGR and the two different phenotypes. The theory supporting the central role of the placenta in the genesis of FGR has the limitation of considering the placenta as a single item. However, its functioning depends on adequate systemic maternal perfusion; thus, maternal cardiovascular function is a relevant factor to acknowledge .In recent years, there has been a growing interest on the possible relationship between FGR and inadequate maternal systemic hemodynamic adaptations to the pregnancy.It is well known that maternal cardiovascular system physiologically changes during pregnancy. The major hemodynamic adaptations include increased cardiac output (CO), expanded blood volume, and reduced systemic vascular resistance (SVR) and blood pressure. SVR progressively drops from the first trimester and nadirs in the mid-second trimester ending in a plateau . CO incrDuring physiological pregnancy, a strict interaction occurs between placental development and maternal systemic adaptation to the pregnancy Fig.  15]: th: th15]: Pregnancy is a challenge for women’s cardiovascular system, acting as a medical stress test for the mother . EchocarSeveral studies have investigated the possible relationship between impaired hemodynamic adaptation during gestation, adverse pregnancy outcomes (APOs), and maternal cardiovascular diseases (CVD). A retrospective population study (CHAMPS study)  performeIn a recent review Lane-Cordova et al. summarized the role of APOs in the pathogenesis of later CVD . They coConsidering the different clinical phenotypes of early and late FGR and the heterogeneity of placental histology, this review focalizes on the possible role of maternal cardiac function in the genesis of both early and late-onset FGR.According to Delphi consensus, the diagnostic criteria for early FGR are fetal abdominal circumference (AC) or estimated fetal weight (EFW) < 3nd centile or umbilical artery absent end diastolic flow (UA-AEDF) which are detected before 32 weeks of gestation. Alternatively, early FGR can be diagnosed if there are at least two of the subsequent three criteria: AC/EFW < 10th centile, uterine artery pulsatility index (UtA-PI) > 95th centile, and UA-PI > 95th centile [The reported incidence of early FGR is about 0.5–1%. This condition is commonly associated with preeclampsia (PE) (60–70%) and abnormal placentation; it often results in poor fetal/neonatal outcomes, including perinatal mortality .Defective placentation, due to a poor trophoblastic invasion of the maternal spiral arteries, is believed to play a central role in the pathogenesis of early-onset PE and FGR. The “placental theory” suggests that FGR may develop as a consequence of abnormal placentation with altered trophoblast invasion . A failuThe retention of the smooth muscular cells in the spiral artery walls may also cause intermittent perfusion leading to a stress response in the placenta. The stress related to placental malperfusion is likely to be an oxidative stress, secondary to the process of ischemia-reperfusion of intervillous space. It causes the release of a number of mediator factors, including pro-inflammatory cytokines, exosomes, and cell-free fetal DNA, into the maternal circulation, resulting in a maternal endothelial cells dysfunction, a systemic inflammatory response, and the clinical syndrome of PE , 28, incHowever, all these circulating placental mediators and the placental theory alone do not explain the correlation between APOs and maternal cardiovascular diseases. In particular, the placental theory does not explain why women who had PE in their pregnancies have a higher cardiovascular risk later in life or why women with pre-pregnancy cardiovascular risk factors have a higher risk of PE and FGR in pregnancy .There are two not mutually exclusive hypotheses associating placental abnormalities and maternal cardiovascular system adaptation in early FGR. The first one places the placenta as the primary cause of hemodynamic changes. High placental vascular resistance increases maternal uterine artery impedance, which contributes to an increase in maternal peripheral vascular resistance. This increase in maternal cardiac afterload opposes maternal cardiac output . TherefoThe second hypothesis supports the role of a pre-pregnancy low maternal CO/high SVR causing reduced placental perfusion. This results in trophoblast impairment and triggers the consequences on the fetal and maternal side of the placenta. This sequence is compatible with the idea of maternal cardiovascular rather than primary placental dysfunction being the origin of complicated pregnancy . This hyActually, the two hypotheses probably coexist Fig. . The perMoreover, it has been widely demonstrated that chronic hypertension is a risk factor for subsequent development of placenta-mediated complications such as FGR, superimposed PE, severe hypertension, and preterm birth (PTB) , 54. DifOther recent findings have led to the re-evaluation of the cause-effect inference between trophoblast development and spiral artery transformation, focusing on the role of maternal systemic hemodynamic rather than local placental development. It is well known that uterine artery blood flow Doppler assessment is a useful tool for the prediction of early-onset PE and FGR. The relationship between uterine artery blood flow and trophoblast cell function is considered to be a proxy of trophoblast invasion, indicating that the persistence of high resistance in the uterine artery Doppler indices reflects an impaired trophoblast invasion and inadequate spiral artery remodeling. A modern point of view suggests an inverse biological association: maternal poor hemodynamic adaptation to pregnancy leads to increased uterine resistance and, in turn, poor placental perfusion may result in impaired trophoblast invasion and function .Late FGR is diagnosed after 32 weeks of gestation by an AC/EFW below the 3rd centile or by at least two of the following three criteria: AC/EFW < 10th centile, AC/EFW centile crossing more than two quartiles on growth curves, cerebroplacental ratio (CPR) < 5th centile, or UA-PI >95th centile [Late FGR is rarely associated with other placental disease such as PE (15%) and is related with a low perinatal mortality rate. However, this is a more frequent condition with an estimated prevalence of 5–10% and its detection and diagnosis are still a challenge for physicians. Therefore, late FGR has a significant clinical impact in terms of fetal outcomes .Unlike the early phenotype, late FGR does not seem to be primarily determined by abnormal placentation in the first trimester . HoweverSupporting this hypothesis, neither early assessment of placental function nor second-trimester uterine artery Doppler velocimetry screening in the general population has the ability to predict late-onset FGR. Uterine artery Doppler impedance classically is thought to represent placental development through spiral artery invasion: high impedance reflects inadequate trophoblast invasion and narrow spiral arteries. It is a useful screening for early-onset PE and FGR, but not for late-onset phenotypes.Accordingly, in 2016, a multicenter randomized trial among 11,667 pregnant women demonstrated that routine second-trimester uterine artery Doppler ultrasound in non-selected population has low sensitivity in detection of late FGR (24%) and SGA fetuses (18%) .Moreover, it has been suggested that the hypoxic status observed in the placenta of late FGR is not related to defective trophoblast invasion but it is mainly determined by subsequent intervillous malperfusion and reduced intraplacental oxygen concentration –67. IncrThe placenta of late-onset FGR may also show completely different late-onset histological findings with delayed villous maturation. This is a placental histopathologic finding that can be found near the end of pregnancy and rarely before 34 weeks of gestation . It is cAll considered, it is challenging to identify methods for the early prediction of late-onset FGR and there is no strong evidence of the best approach for its detection and management .As previously stated, Doppler velocimetry assessment of uterine arteries during the first trimester of pregnancy is not a sensitive method for predicting the risk of late-onset FGR. Recently, Binder et al.  evaluateIt has been suggested that pregnancy may unmask a preexisting sub-clinical maternal cardiac dysfunction. Thus, a subclinical impairment of maternal cardiac function existing before pregnancy may emerge during the strain of pregnancy, failing the adaptation of maternal cardiovascular system to the feto-placental demands , 71. TheIn a prospective observational cohort study, Stott et al.  assessedTherefore, assessment of maternal hemodynamic rather than uterine artery Doppler in the second trimester could improve to identify those women at higher risk to develop late FGR. Using multivariate logistic regression analysis, Roberts L.A. et al. demonstrated that prediction of birthweight < 3rd percentile is improved by assessment of maternal hemodynamic function along with maternal history and demographics, fetal biometry, and Dopplers . EspeciaThe maternal hemodynamic variables, longitudinally assessed in late FGR and adequate for gestational age (AGA) fetuses, differed from the first to the third trimester of gestation. Moreover, the highest difference in cardiovascular parameters was at 25 weeks , suggestBased on these evidences, the maternal cardiovascular system in late-onset FGR becomes unable from the second trimester onwards to respond to the increasing demands of pregnancy, when the placenta, normally developed until that moment, increases its requests. The hypoperfusion and maladaptive response of the placenta could be the consequence of a primary pump deficit, which leads to an altered maturation of the placental villous tree and therefore to an altered fetal growth , 75 Fig.Fig. 3PaMaternal hemodynamic indices are relevant both for screening and diagnosis of FGR, helping to distinguish between a small for gestational age (SGA) and a growth-restricted fetus , 45.In a recent prospective study , a singlConsistent results are derived from a retrospective study among 51 pregnancies with FGR or SGA fetuses . Women wAs the pre-pregnancy cardiac function is related to the placenta function, so does the placenta with the post-pregnancy cardiovascular state. It has been suggested that defective placentation and the resultant proinflammatory state are characterized by an antiangiogenic state and endothelial and cardiac maternal dysfunction; this persists in the postpartum and may underlie development of CVD later in life. In fact, maternal cardiac dysfunction was also found in non-pregnant women with a history of PE and/or FGR. Moreover, in these women, a new pregnancy might worsen an impaired hemodynamic environment, which can predispose to the recurrence of similar complications .In 2016, Valensise et al. found signs of systolic and diastolic dysfunction in the preconception state before a second pregnancy complicated by recurrent early PE. Moreover, previous early preeclamptic patients with non-recurrent PE showed left ventricular structural and functional features intermediate compared to controls and recurrent PE .A large Norwegian register–based study  reportedGestational age at delivery and fetal growth seem to correlate with subsequent risk of maternal cardiovascular disease regardless of PE, with a risk of coronary heart disease that is markedly increased in women with most serious and early FGR cases .In addition, endothelial dysfunction in the postpartum state, demonstrated by impaired endothelium-dependent vasodilatation and increased arterial stiffness, was found in women with previous early-onset PE and FGR, but not in women with previous late-onset PE , 81. A hFrom a clinical point of view, some FGR-linked histological lesions of decidual arteriopathy share similar aspects with CVD, representative of systemic and local inflammatory milieu. During pregnancy, a fine maternal immune system regulation is necessary to control fetal growth  and to aThis represents a vicious circle: pre-pregnancy cardiovascular risk factors, together with innate immune dysregulation and genetic predisposition, may contribute to poor placentation. Incomplete placentation results in placental ischemia and release of antiangiogenic and proinflammatory factors causing cardiovascular damage during pregnancy and in post-partum period .There are no proven treatments for FGR able to improve fetal growth in utero. The only effective therapeutic option is timely iatrogenic PTB after administration of maternal corticosteroids and magnesium sulphate to improve neonatal outcome .Several studies showed that the prophylactic use of low-dose aspirin in women at high risk of developing PE and FGR may reduce the prevalence of these complications , 89. AspThe understanding of the possible role of maternal hemodynamics in the pathogenesis of FGR may suggest the development of new therapeutic tools based on this mechanism.l-arginine by a family of calcium-calmodulin-dependent enzymes called nitric oxide synthases (NOS). It is released by endothelial cells with a physiological vasodilating effect that increases blood flow. Moreover, it induces a decreased responsiveness to vasopressors and inhibition of platelet function [NO donors have been widely studied for both prevention  and manafunction . As prevfunction . NO donofunction  found thfunction  confirmePromising results have been recently obtained in case of statin administration. Statins are generally used for the inhibition of cholesterol biosynthesis and for the prevention of atherosclerotic cardiovascular diseases. Treatment with pravastatin 40 mg/die in pregnancies affected by early-onset FGR was associated with a significantly better angiogenic profile, improving the sFlit1/PlGf ratio . This reRecently, sildenafil, a phosphodiesterase type 5 inhibitor, has been proposed for the treatment of FGR , 101. SiThe identification of maternal hemodynamic status should be helpful in choosing the most appropriate treatment in women with hypertension and FGR, according to the underlying cardiovascular phenotype. In those cases, drugs with beta-blocking activity are negatively chronotropic and reduce CO. Therefore, they may be harmful in women with FGR in which the uteroplacental circulation is already impaired. Conversely, calcium antagonists are likely to be more effective in the high-SVR state that characterizes hypertensive women with FGR .Pregnancy represents a challenge to women’s cardiovascular system, acting as a medical stress test for the mother.  This preAs far as recent evidence suggests, both early and late FGRs are associated with placental disease, albeit with different pathways. In the early-onset FGR, the placental abnormal development seems to be the cause of FGR and is associated with histological signs of abnormal early implantation . In the late-onset FGR, it is unclear whether placental abnormalities are related to less severe forms of abnormal placentation at early pregnancy or to late placental damage occurring during the second half of pregnancy .The new interesting point of view includes the interaction between placental and maternal cardiovascular system in the pathogenesis of FGR Fig. .Fig. 4OrIt has been hypothesized that early-onset PE and FGR are due to an association between an intrinsic placental dysfunction with a pre-existent maladaptation of the maternal cardiovascular system. Instead, late-onset fetal growth disorders are more likely to be associated with an acquired placental dysfunction as a result of a static hemodynamic system not being able to meet the excessive demands of an advanced or overgrown pregnancy , 72.A better understanding of maternal hemodynamics in pregnancies complicated by FGR could bring various benefits in clinical practice. In fact, it could have potential therapeutic implications, improve screening tools, help distinguish SGA from FGR, and prevent future CVD in these women. However, further studies are needed to verify if the changes in hemodynamic parameters among FGR women are already present in pre-gestational period and to examine the possibility of manipulating maternal hemodynamics to improve pregnancy outcomes.


The pharmacological elimination of senescent cells by the means of selectively toxic compounds, termed senolytics, is a strategy that has gained increasing interest in the last ten years as a potential means to alleviate age-related diseases, as well as to potentiate chemotherapy and to prevent malignant transformation. Senescence is a cellular response to stress, alternative to apoptosis, in which the cell undergoes a quasi-irreversible proliferative arrest accompanied by important modifications of its transcriptional programs, and often secretes a collection of inflammatory factors termed the senescence-associated secretory phenotype (SASP). Senescence-inducing stressors are various and notably include telomere attrition, chemotherapeutic agents, ionizing radiations, oxidative stress, and oncogene activation. Senescent cells accumulate during aging and in many pathological contexts, where they play a detrimental role often owing to SASP-mediated chronic inflammation. In many pathological conditions in which accumulating senescent cells are implicated, their elimination by senolytics was shown to be highly beneficial.The oncogenic V600E mutation in the BRAF kinase induces senescence in melanocytes and leads to the formation of nevi (moles). Melanoma can arise from BRAF-V600E melanocytic nevi, suggesting mechanisms of senescence escape at play. One strategy to prevent melanoma formation in patients at risk would be to eliminate pre-malignant, senescent melanocytes, with senolytics.In a recent study, we sought to uncover novel vulnerabilities of BRAF-V600E-induced senescent cells (BRafSen cells) that could be targeted by senolytics to prevent melanoma formation . To thisThe relationship between cellular senescence and autophagy is regarded as paradoxical. Autophagy activation in response to stress can successfully resolve it and thus spare the cell from entering senescence. Aiming to identify ways of specifically eliminating cancer cells, Schepers et al. recently showed that targeting macroautophagy by ULK1 inhibition induces senescence in a panel of cancer cells , and renders cells sensitive to senolysis by BH3-mimetic ABT-263 . On the Beyond bulk autophagy flux modulations to cope with increased secretory demands, finer processes appear to be at play in regulating proteostasis in senescence. Recently, it was shown that the stability of a defined set of proteins was regulated by selective autophagy in senescence through differential interactions with ATG8 family receptors. This selective autophagy network was fundamental in shaping several facets of the senescent phenotype, including redox homeostasis (through KEAP1 degradation), SASP production (through TNIP1 degradation), and proteostasis (through eIF3 degradation) . In anot


Bacteria possess the ability to adapt to changing environments. To enable this, cells use reversible post-translational modifications on key proteins to modulate their behavior, metabolism, defense mechanisms and adaptation of bacteria to stress. In this review, we focus on bacterial protein switches that are activated during exposure to oxidative stress. Such protein switches are triggered by either exogenous reactive oxygen species (ROS) or endogenous ROS generated as by-products of the aerobic lifestyle. Both thiol switches and metal centers have been shown to be the primary targets of ROS. Cells take advantage of such reactivity to use these reactive sites as redox sensors to detect and combat oxidative stress conditions. This in turn may induce expression of genes involved in antioxidant strategies and thus protect the proteome against stress conditions. We further describe the well-characterized mechanism of selected proteins that are regulated by redox switches. We highlight the diversity of mechanisms and functions (as well as common features) across different switches, while also presenting integrative methodologies used in discovering new members of this family. Finally, we point to future challenges in this field, both in uncovering new types of switches, as well as defining novel additional functions. Most bacterial cells live in a dynamically fluctuating environment, requiring rapid responses to enable successful growth. These changing environments might induce stress conditions , which challenge bacterial homeostasis and macromolecules, affecting a wide variety of cellular processes. Thus, it is not surprising that bacteria and other organisms evolved different sensors and first line of defense mechanisms to combat environmental assaults. One such stress-response strategy utilizes rapid post-translational modifications of proteins that induce the general response and trigger defense activities. Reversible post-translational modification of proteins is one of the major toolboxes available to cells, which ensures a plasticity of the cellular proteome, as well as rapid control of diverse cellular functions, including stress specificity. While phosphorylation is one of the major regulators of the cell cycle , oxidatiProkaryotes constantly produce reactive oxygen species, ROS,  during their life cycle as a consequence of growth in an aerobic environment . These RAbnormal levels of oxidative stress can cause irreversible damage to diverse cellular macromolecules including nucleotides, lipids and proteins, affecting their function and stability. The exposure of bacteria to harmful oxidation has led to the evolution of extensive damage repair systems which consist of a large repertoire of antioxidant enzymes that detoxify different oxidants and convert them into harmless molecules . The maiNon-specific protein oxidation might lead to various post-translational modifications of sulfur-containing residues (Cys and Met) and aromatic residues , as well as induce undesirable disulfide bonds and affect protein cofactors, especially metal centers – all which might lead to protein inactivation, unfolding, accumulation of toxic aggregates and even cell death .Escherichia coli with low levels of peroxide increases its survival during severe oxidative stress conditions, emphasizing the importance of dynamic responses and bacterial adaptation to changing intracellular ROS levels. Moreover, it was shown that production of intracellular ROS might provide antibiotic tolerance in Mycobacteria, suggesting a tight regulation between redox homeostasis and adaptation pathways , to detoxify undesirable levels of ROS  and to protect the cellular proteome against potential damage .One of the main strategies of this defense system is to utilize rapid and reversible oxidation-dependent modification of specific protein thiol residues, serving as redox-sensitive switches of the defense proteins and mediating their rapid activation .Another strategy – which can be coupled to modification of the thiol groups – is exploiting redox properties of metal centers to regulate proteins during fluctuating oxidant levels. Thus, bacteria (and eukaryotes) have developed an elegant way to convert “protein weakness” into a powerful and robust mechanism to regulate the expression of genes that provide a defense against oxidative stress. They are then able to detoxify ROS using reversible reduction-oxidation cycles of catalytic cysteine residues or cofactors, restore the redox status of proteins and maintain protein quality control under stress conditions. Here, we will briefly discuss different types of protein switches and their working mechanism, which enable bacteria to adapt and defeat oxidation-related challenges during their life cycle.2O2), hydroxyl (·OH) and superoxide anion (O−2) radicals. Accumulation of these ROS results in negatively charged modification of reactive protein thiols, in the form of sulfenic (-RSOH) or sulfinic acids (-RSO2H), or in the formation of non-native, covalent disulfide bonds within and between different proteins , which undergoes sulfenylation (S-OH) and consequent disulfide formation with the adjacent cysteine (Cys 208 in E. coli)  . DisulfiE. coli)  (FuangthE. coli)  InterestThis ultimately results in different DNA binding affinity and specificity, , dependiBacillus subtilis  on Cys 15 and small local structural changes (2H) or sulfonic (-RSO3) acid leads to the detachment of OhrR from the ohrA operator region  are thioredoxin and glutaredoxin enzymes that restore the redox status of proteins using reduction-oxidation cycles of their conserved catalytic cysteine residues with the help of cellular cofactors, such as NADH, NADPH, and Glutathione . NumerouMetalloproteins are central actors in a wide number of biological processes . The chevia a Fenton reaction , (3+ ions as well as a superoxide ion (O2−), which is further converted to peroxide and water -CorE binds to DNA whereas Cu+-bound CorE does not , which is activated under conditions that lead to protein misfolding and accumulation of toxic aggregates, such as oxidative unfolding. Hsp33 “senses” the presence of oxidants or chlorine species  through a highly reactive Zn center, comprising of four completely conserved cysteines forming CXCX and CXXC motifs harboring one Znced form . Oxidaticed form . Oxidaticed form , destabiced form  , oteomics .During the last decade, several studies showed that antibiotic treatment alters redox homeostasis and leads to the accumulation of ROS in bacteria, which might be an additional cause for cell death . While tin vivo or in vitro, in order to detect changes in the redox status of either single or multiple proteins in a biological sample  and X-Ray crystallography] were able to define the exact redox cascade mechanism in metalloproteins and define catalysis at the atomic level. This is challenging in the case of redox switch proteins, which require structural plasticity or oligomerization  for their activity, which complicate the obtention of an atomic structure using NMR or X-Ray. In this case, structural mass spectrometry  takes on its undebated role. HDX-MS analysis of Hsp33’s working cycle has enabled mapping of redox-dependent conformational changes on both the chaperone and its substrate, mediating substrate binding and release .in vitro on purified systems. The recent development of in cell-NMR and in cell-EPR will give a better picture of in vivo metal-switch reactions. In addition, finding new family members might be possible with the development of metallomics approaches combined with spectroscopy or other tools, to find redox-regulated metal centers.Furthermore, research on redox-regulating metalloproteins sits in the junction between structural biology and chemistry. During the last decade, biophysical approaches such as UV-Visible, EPR (electron paramagnetic resonance), NMR or X-ray absorption spectroscopy have pushed the metal-switch field forward, providing high resolution mechanisms of enzymes and transcriptional factors. However, to date, metal-switch proteins have been mainly described However, despite the many fascinating breakthroughs that have been made over the past several years, we need to develop a new repertoire of methodologies addressing non-thiol regulation. Recently, a few technologies were established to investigate methionine and tyrosine oxidation, however, more should be done in this field. The development of these methodologies will open a door to uncover new types of switch proteins, employing other regulatory sites and chemistry.Another aspect that should be addressed in the redox-switch protein research is the multi-functionality of this class of proteins. It is clear that many redox-regulated proteins cannot be simply defined by loss-gain of function under oxidation-reduction conditions. Many of the redox-regulated proteins have more than one biological function and specificity to different radicals. One of the next challenges in the field is to understand the evolutionary path of redox switch proteins, their multiple functionality, and their reactivity.


Glioblastoma is a primary malignant brain tumor with a median survival under 2 years. The poor prognosis glioblastoma caries is largely due to cellular invasion, which enables escape from resection, and drives inevitable recurrence. While most studies to date have focused on pathways that enhance the invasiveness of tumor cells in the brain microenvironment as the primary driving forces behind GBM’s ability to invade adjacent tissues, more recent studies have identified a role for adaptations in cellular metabolism in GBM invasion. Metabolic reprogramming allows invasive cells to generate the energy necessary for colonizing surrounding brain tissue and adapt to new microenvironments with unique nutrient and oxygen availability. Historically, enhanced glycolysis, even in the presence of oxygen (the Warburg effect) has dominated glioblastoma research with respect to tumor metabolism. More recent global profiling experiments, however, have identified roles for lipid, amino acid, and nucleotide metabolism in tumor growth and invasion. A thorough understanding of the metabolic traits that define invasive GBM cells may provide novel therapeutic targets for this devastating disease. In this review, we focus on metabolic alterations that have been characterized in glioblastoma, the dynamic nature of tumor metabolism and how it is shaped by interaction with the brain microenvironment, and how metabolic reprogramming generates vulnerabilities that may be ripe for exploitation. Glioblastoma (GBM) is a primary malignant brain tumor with a median survival under 2 years . The pooClinical observations and experimental studies have led to numerous hypotheses attempting to define the factors which drive tumor invasion, but whether these changes are causative is widely contested . RegardlSubsequent studies have revealed differentially expressed genes at individual steps in the process of cellular invasion . The expMore recently, attention has been given to the notion that invasive cells also require specific metabolic traits to survive and grow in new environments which substantially vary in their nutrient and oxygen availability from the tumor core . Such meCellular growth and division depend on the uptake of nutrients from the surrounding environment, which are in turn metabolized to produce energy and maintain cellular homeostasis . The braHigh glucose levels have been linked to increased tumor invasion and poor patient survival . To gainIn neurons and glia alike, glucose is metabolized via glycolysis into pyruvate, which then enter the tricarboxylic acid (TCA) cycle to generate ATP through oxidative phosphorylation (OXPHOS) . In contin vitro and metastasis in vivo  is a cytosolic enzyme that catalyzes the conversion of glucose-6-phosphate into fructose-6-phosphate in the second step of glycolysis . PGI is  in vivo . Fructos in vivo . Loss of in vivo . Pyruvat in vivo . In colo in vivo .The Warburg effect is well studied in glioblastoma . Like maWhen metabolizing glucose, invading tumor cells must also divert carbon from glycolysis into the pentose phosphate pathway (PPP) for nucleotide synthesis and to combat oxidative stress . The PPPThe oxidative arm of the PPP utilizes G6P as its substrate and leads to the generation of NADPH. The non-oxidative reactions of the PPP lead to the generation of ribose-5-phosphate (R5P) for nucleotide biosynthesis . Many glTo meet GBM’s high metabolic demands, glucose uptake is increased through the upregulation of glucose transport proteins . ExpressLipids are a diverse group of water-insoluble molecules essential for numerous biological processes . Their fde novo biosynthesis by astrocytes and delivered to neurons within high-density lipoproteins containing apolipoprotein E (Apo-E)  . Unlike  (Apo-E) , the met (Apo-E) . Studies (Apo-E) .In addition to increased extracellular cholesterol uptake, GBM cells exhibit dysregulated cholesterol efflux and synthesis . At highLipidomics studies have shown the presence of triglycerides in two biopsies from glioblastomas (GBM) that had no treatment, but absent in healthy adult brain. Given that triglycerides contain three fatty acids and act as energy storage in addition to unsaturated fatty acids prominent in high-grade intracranial tumors, it appears that gliomas have developed an altered metabolism for fatty acids . Triglycin vitro and in xenograft mouse models (Fatty acid (FA) synthesis and catabolism are also among the lipid metabolic pathways with demonstrated alteration in Glioblastoma, with paradoxical elevation observed in both pathways. Fatty acids are able to cross the BBB and are readily available at the tumor’s invasive front . The Fate models . Global e models . The simOn a mechanistic level, expression of the FA uptake channel CD36, is upregulated under hypoxia in several types of cancer , 36. CD3Amino acids are increasingly recognized as important fuels for supporting cancer growth and division . Biosyntin vitro studies , which facilitate epithelial tumor cell invasion . CAFs anIn vitro studies have shown restriction of the essential amino acid methionine slows GBM cell in vitro via inhibition of key oncologic signaling proteins including PI3K, p38MAPK, and ERK  is the central point of convergence for intermediates generated in the metabolic pathways altered in GBM . CarbohyInterestingly, it has been primarily genomic, rather than metabolic studies that have shed light on the importance of oxidative phosphorylation in GBM . SeveralIDH mutant tumors constitute roughly 10 percent of all glioblastomas . Tumors +-dependent enzymes that interconvert isocitrate and α-ketoglutarate (αKG) in cytosol and mitochondria, respectively. Genome-wide exon-sequencing studies of gliomas have revealed IDH1 R132H activating point mutations in as many as 80–90% of low-grade gliomas (LGGs) (IDH1 and IDH2 are NADPs (LGGs) . Mutatios (LGGs) . The pris (LGGs) . Consequs (LGGs) .Mutations in additional TCA and TCA adjacent enzymes are found in GBM with lower frequency and comparatively fewer known functional implications compared to IDH . NotablyThe brain extracellular matrix (ECM) occupies a notable portion of the CNS and contributes to its normal physiology . It is pAlterations to the ECM occur in many diseases of the CNS, including glioblastoma . As GBM During the invasive process, GBM cells become polarized, and their outer edge undergoes dynamic cytoskeletal rearrangements that facilitate adhesion to the adjacent ECM. This process is generally believed to be regulated by receptors in the integrin protein family . Integriin vitro studies  are proteases believed to play a central role in GBM invasion, owed to their ability to degrade many brain ECM components . This faUrokinase-type plasminogen activator (uPA) is a serine protease which, along with its receptor uPAR, plays a role in invasion and neovascularization in gliomas . MoleculGlioblastoma Stem Cells (CSCs) constitute a small percentage of GBM tumor cells and demonstrate two principal features of stem cells: self-renewal and differentiation . GSCs arDistinct microenvironments in GBM tumors; including the hypoxic core, the perivascular niche, and the invasive tumor edge, add to the heterogeneity and the dynamic behavior of GSCs in these tumors . GSCs hoThese dynamic metabolic changes in GSCs create a challenge when selecting therapeutic targets, and warrants exploration of targeting multiple pathways simultaneously. For example, radiation targets highly proliferative cells sparing slow cycling cells . TherefoTo invade adjacent brain tissue successfully, GBM cells must avoid detection and defend themselves from the host immune system. The immune composition of GBM’s microenvironment evolves with tumor stage but is predominantly comprised of immune suppressive cells . There aFollowing activation, T cells and DC undergo rapid expansion, with an accompanying increase in bioenergetic demand . These aThis preference for glycolysis has also been demonstrated in other immune cells including macrophages, neutrophils, B cells and natural killer (NK) cells . HoweverGlioma cells also influence their adjacent immune microenvironment through amino acid metabolic pathways via increased uptake of branched chain amino acids (BCAAs) . GlioblaFurthermore, GBM tumors promote macrophage and T cell dysfunction through expression of the ectonucleosides CD39 and CD73 . These iMoreover, hypoxic conditions associated with the GBM tumor microenvironment can also contribute to immune system suppression . HypoxiaIn summary, tumor metabolism plays a crucial role in negating local immune detection and response. This dynamic interplay must be taken into consideration when targeting invasion in GBM.The metabolic divergence between GBM cells and normal astrocytes holds potential for the discovery of novel therapeutic targets . MetabolCurrent standard treatment for GBM involves surgical resection followed by concurrent radio and chemotherapy . DespiteWhole brain radiation has previously been shown to induce metabolic changes in GBM cells . UntargeSeveral studies have reported the metabolic changes associated with Temozolomide (TMZ) resistance. GBM treatment with TMZ induces changes in mitochondrial complex activity and glutamate metabolism . TMZ resThe VEGF inhibitor bevacizumab does not improve patient overall survival and has shown to be associated with increased invasion of GBM cells . VEGF inAmong the metabolic pathways targeted in GBM by researchers, glycolysis has historically been the most prevalent . Most meThe results of the aforementioned trials suggest the ketogenic diet can be safely implemented in patients and can achieve sufficient levels of ketogenesis . Patientin vitro and in vivo with small molecules that disrupt key steps in crucial metabolic pathways. 2-deoxyglucose (2DG), which halts glycolysis following its initial phosphorylation by hexokinase, has been shown to inhibit GBM growth to a greater degree when combined with targeted radiation than radiation alone, and was safely used in a clinical trial in India with modest improvements in survival and reported patient quality of life (in vitro. 3-bromo-2-oxopropionate-1-propyl ester (3-BrOP), an inhibitor of hexokinase (HK) and 3-phospho dehydrogenase (3-PD) targets GSCs and therapeutic resistance acquired by GSCs. Combination treatment of 3-BrOP with carmustine has been shown to have a synergistic effect on decreased tumor formation  have come into focus as potential therapeutic targets to mediate the metabolic shifts in GBM due to their ability to regulate levels of specific metabolic genes of interest . Example3) which inhibits cytoplasmic phospholipase A2 in phospholipid metabolism, and A922500, which inhibits diacylglycerol-acyltransferase 1 (DGAT1) in the formation of lipid droplets from FFAs, are two more targets that the inhibition of which has stunted tumor growth in intracranial xenograft mouse models (Long overlooked in favor of other pathways, lipid metabolism has increasingly come into focus in the targeting of metabolic pathways in GBM. LXR-623 and archazolid B are two promising metabolic therapies and inhibit cholesterol uptake and recycling, respectively. One or both drugs can potentially be used to target cholesterol metabolism which has been linked to GBM’s invasive spread . Arachide models , p. 1.in vivo (in vivo. Combination treatments using Phenformin and TMZ inhibit tumor growth in vitro and in vivo (in vitro and in vivo (Altering mitochondrial activity holds promise for targeting metabolism in GBM. Nigericin, a drug identified in a small molecule screen was able to induce mTOR inactivation, increase autophagy and decrease tumor growth in vivo . Metformin vivo . Further in vivo . An arse in vivo . Recentl in vivo . FurtherWhile therapies targeting metabolic pathways to reduce invasion in GBM have shown tremendous promise, there are multifactorial challenges in utilizing these treatments in patients. The suppression of invasion via inhibition of cellular metabolism in GBM is subject to the same difficulties faced by pharmacologic treatments of any disease of the central nervous system, that is, penetration of the BBB . FurtherAlthough potential therapies targeting tumor metabolism hold promise in the treatment of invasive GBM, several challenges in the drug development pipeline exist . Drugs dMetformin, an example of a repurposed drug with growing interest for its potential to target tumor metabolism, is a first-line treatment for type II diabetes . Metformin vitro studies and observational research in patient cohorts (in vitro (Another commonly used drug group, statins, have been implicated for potential repurposing to treat glioma based on promising  cohorts . Statins cohorts . These din vitro . Existinin vitro .Additional drugs that have been repurposed to stymie invasion in GBM include, NSAIDS, Disulfiram, and Ritonvir. These act through COX-2 inhibition and downstream reduction of c-myc and LDH expression, inhibition if ALDH and oxphos, and disruption of glutamine metabolism, respectively . The eviThe collective body of knowledge concerning the metabolic profile of invasive glioblastoma is rapidly evolving. The study of tumor metabolism is shifting from a static picture shaped by genetic mutations or environment alone to a dynamic view in which genotype and microenvironment interact to form the metabolic profile of invasive tumor cells, exposing potential vulnerabilities that are ripe for therapeutic exploitation. Research in this area is challenging because it is not amenable tracking data at any particular point in time, but rather requires a nuanced understanding of physiologic and biochemical alterations in flux, especially in the dynamic environments which brain tumors encounter. Fortunately, novel clinical and research tools are providing powerful insights. By integrating the full spectrum of system-wide, unbiased “omics” screens available to researchers, and cutting edge diagnostic clinical tools including radiotracer and flux imaging technologies, a new understanding of the molecular basis of glioblastoma metabolism has begun to emerge, including metabolic vulnerabilities that could be targeted therapeutically. Additionally, the development of integrative models based on tumor molecular markers, microenvironment, or both are continuously revealing additional therapeutic targets. This review has focused primarily on the actionable targets and routes of exploration that will be the most useful for finding them, with the goal of expanding the breadth of pharmacologic interventions through new drug development and repurposing. Glioblastoma research has been at the forefront of cancer research for the past 10 years, including the elucidation of critical mechanisms by which tumors undergo metabolic reprogramming. It is time for this research to translate to the clinical setting for the benefit of patients.JG prepared the figures and table. All authors contributed to the conception, drafting, and revising of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ABSTRACT IMPACT: With a growing number of Americans enrolled in high-deductible health plans, patients, especially those with chronic conditions, face increasing cost-sharing burden. We aim to develop a novel behavioral intervention to help patients use consumer strategies to better manage their health care spending. OBJECTIVES/GOALS: To assess patient preferences to develop an intervention to encourage the use of cost-conscious strategies to manage out-of-pocket health care spending among high-deductible health plan (HDHP) enrollees with chronic conditions. METHODS/STUDY POPULATION: This mixed-methods study is first conducting semi-structured telephone interviews of up to 20 adults with one or more chronic conditions who are enrolled in an HDHP. Preliminary findings from these interviews are being used to inform the design of a national internet panel survey of at least 300 HDHP enrollees. Collectively, the interviews and survey will assess experiences of HDHP enrollees and their preferences for the content, design, format, and mode of an intervention to help them engage in cost-conscious health care behaviors. These findings will then be used to develop a novel behavioral intervention that will subsequently be pilot tested for acceptability, feasibility, and preliminary efficacy. RESULTS/ANTICIPATED RESULTS: Early interview data identified gaps in knowledge of health care consumer strategies among HDHP enrollees with low confidence in being able to engage in cost-conscious health care behaviors. Several participants indicated interest in an intervention to learn more about how to engage in cost-conscious strategies . Most early interview participants preferred an easily accessible technological intervention, such as a website or app. Interviews are continuing, and the national survey will be fielded in early 2021. DISCUSSION/SIGNIFICANCE OF FINDINGS: HDHP enrollees with chronic conditions could benefit from an intervention that helps them manage their high cost-sharing. Based on the results of interviews and a national survey, we will develop and pilot test a novel behavioral intervention to promote use of cost-conscious health care behaviors.


A large number of neurological disorders can affect renal transplant recipients, potentially leading to disabling or life-threatening complications. Prevention, early diagnosis and appropriate management of these conditions are critical to avoid irreversible lesions. A pivotal role in the pathogenesis of common post-transplant neurological disorders is played by immunosuppressive therapy. The most frequently administered regimen consists of triple immunosuppression, which comprises a calcineurin inhibitor (CNI), a purine synthesis inhibitor and glucocorticoids. Some of these immunosuppressive drugs may lead to neurological signs and symptoms through direct neurotoxic effects, and all of them may be responsible for the development of tumors or opportunistic infections. In this review, after a brief summary of neurotoxic pathogenetic mechanisms encompassing recent advances in the field, we focus on the clinical presentation of more common and severe immunosuppression-related neurological complications, classifying them by characteristics of urgency and anatomic site. Our goal is to provide a general framework that addresses such clinical issues with a multidisciplinary approach, as these conditions require. A wide array of neurological complications may impact on the outcome of renal transplant recipients, possibly leading to disabling or life-threatening diseases. Some of these complications are caused by the disorders that led to transplantation and by dialysis . Since cThe most crucial role in the pathogenesis of post-transplant neurological disorders is played by immunosuppressive drugs. The choice of immunosuppressive regimens varies widely across transplant programs, nevertheless, the most frequently adopted schedule rests on triple immunosuppression comprising a calcineurin inhibitor (CNI) such as tacrolimus (TAC) or cyclosporine (CsA), a purine synthesis inhibitor, such as mycophenolate (MPA) or azathioprine, and glucocorticoids. Some centers have been gaining experience with inhibitors of the mammalian target of rapamycin (mTOR) and, more recently, with belatacept. Anti-thymocyte globulins (ATG), anti-CD52 , anti-CD25/IL-2R (basiliximab) and anti-CD20 (rituximab) monoclonal antibodies are also frequently used for induction therapy or treatment of rejection episodes. The proteasome-inhibitor bortezomib has also been used for the management of antibody-mediated rejection in selected cases. Some of these immunosuppressive drugs may exert direct neurotoxic effects, and all of them may be involved in the development of tumors or opportunistic infections [In this review, after a brief overview of neurotoxic pathogenetic mechanisms encompassing recent advances in the field, we focus on the clinical presentation of more common and severe neurological complications. This work deals with the neurological presentations most frequently caused by treatment with immunosuppressive drugs, classifying them by characteristics of urgency and anatomic site  Fig. .Fig. 1NeCalcineurin is a calcium- and calmodulin-binding protein phosphatase that participates in a wide array of cellular processes and calcium-dependent signal transduction pathways, including T-cell activation. CsA and TAC inhibit calcineurin to a similar degree, inducing downstream blockade of IL-2 signaling, ultimately interfering with T-cell activation, proliferation and differentiation. Calcineurin is highly expressed in the central nervous system, particularly in neurons vulnerable to ischemic and traumatic injury.Several mechanisms may be involved in CNI-related toxicity on the central nervous system (CNS) Fig. . CNIs doIn addition to their direct effects on neural cells, CNIs may lead to activation of the major vasoconstriction systems, i.e. the renin–angiotensin and endothelin systems, and increase sympathetic system activity. In addition, CNIs inhibit nitric oxide synthesis and nitric oxide-mediated vasodilation. Altogether, these processes cause vasoconstriction and endothelial dysfunction , which rAzathioprine and mycophenolate interfere with nucleotide synthesis with different mechanisms. Azathioprine is a mercaptopurine prodrug, whose pharmacological activity rests on the formation of two active intracellular nucleotides: thioinosinic acid inhibits the de novo pathway of purine synthesis, while 6-thioguanine interferes with the purine salvage pathway. These actions result in depletion of cellular purine stores and inhibition of DNA/RNA synthesis, hampering lymphocyte proliferation. The immunosuppressive effects of azathioprine are more potent on T cells compared to B cells, owing to intrinsic metabolic differences in these cells. Mycophenolate salts are prodrugs releasing active mycophenolic acid (MPA), which reversibly inhibits inosine monophosphate dehydrogenase and results in a marked reduction of guanosine triphosphate necessary for DNA synthesis. Neurological symptoms directly induced by purine synthesis inhibitors are rare and mild, usually manifesting as depression and headaches. Cases of progressive multifocal leukoencephalopathy (PML), caused by John Cunningham Virus (JCV) infection, have been reported in renal transplant recipients or lupus patients treated with MPA, and thus the Food and Drug Administration warned against this possible risk.Synthetic glucocorticoids exert a wide array of anti-inflammatory and immunosuppressive activities that are mainly mediated by their genomic effects. Glucocorticoids bind to their specific cytosolic receptors, enter the nucleus and either activate response elements that induce anti-inflammatory genes, or repress inflammatory transcription factors (e.g. NF-kB or activator protein-1). These drugs may also produce non-genomic effects with rapid onset and short duration, whose mechanism of action is incompletely understood. At least part of this activity seems to be mediated by membrane receptors that can modulate anti-inflammatory and antioxidant effects .Brain cells express two types of corticosteroid receptors, i.e. mineralocorticoid and glucocorticoid receptors, which differ in distribution and affinity and can exert modulatory effects on a variety of brain functions from early development through late life. The potential effects of glucocorticoids on neuronal activity are determined predominantly by receptor distribution since glucocorticoids easily pass the BBB and virtually reach all brain cells, although local enzymatic conversion and the degree of cell accessibility contribute to the actual intracellular concentration .The use of glucocorticoids in pediatric transplant recipients may be particularly problematic. Exposure to high-dose glucocorticoids in early life can significantly affect the hypothalamic–pituitary–adrenal axis and increase the susceptibility to develop metabolic, neuropsychiatric and neurodegenerative disorders . In addiSirolimus (rapamycin) and its derivate everolimus inhibit the mTORC1 complex, a master regulator of cell growth and metabolism, interfering with signals leading to T-cell proliferation. Both mTOR inhibitors can cross the BBB, but there is considerable uncertainty regarding their potential neuroprotection or neurotoxicity. In the nervous system, the mTOR pathway regulates axonal sprouting, axonal regeneration and myelination, ion channel and receptor expression, as well as dendritic spine growth . mTOR siRituximab is a monoclonal antibody with a high affinity for the CD20 antigen, a membrane protein expressed on B cells. Reports of PML cases in patients treated with Rituximab have raised concerns about its use in renal transplant recipients, since the risk seems to be higher in patients treated with multiple immunosuppressive drugs . HoweverS. pneumoniae, N. meningitidis), which can be however effectively prevented by vaccination against these pathogens.Rabbit ATG are polyclonal antibodies that reduce the number of circulating T cells through cell lysis. Bortezomib, a broad-spectrum proteasome inhibitor, may cause neurotoxic effects through a dysregulation of neurotrophins and blockade of nerve growth factor-mediated neuronal survival induced by NF-κB inhibition. Belatacept is a fusion protein with high affinity for CTLA-4 (CD152), a cell surface protein transiently expressed on T cells that attenuates their activation. This drug blocks T-cell costimulation and may increase the risk of post-transplant cerebral lymphoma in Epstein Barr virus- (EBV) negative patients who receive a kidney from EBV positive donors . FinallySeizures have been reported in 6–36% of transplant recipients  and the Intravenous methylprednisolone pulses (MPP), 500–1000 mg per dose, are the standard treatment for acute rejection and are frequently used for induction therapy. Although MPP therapy may be considered for possible treatment in patients with focal epilepsy, exceptional cases of seizures have been reported after intravenous administration of steroids . ComplicSeveral studies have consistently reported a higher frequency of cerebral vascular events in kidney transplant recipients compared to the general population, with an incidence of 5% in the first year and 9.4% in the second year post-surgery –39. ThisPosterior Reversible Encephalopathy Syndrome (PRES) is a severe neurological complication caused by vasogenic edema, which can lead to permanent deficits when not properly identified and treated . It usuaCentral nervous system infections are extremely frequent in renal transplant recipients, and represent 9–10% of all neurological complications in this population . The subTo narrow down the exceptionally broad differential diagnosis of responsible infectious agents, time from transplantation should always be considered since specific pathogens are more likely to be responsible at different time points Table . In the Six months after transplantation, the infection risk starts to decrease and the most frequently encountered pathogens are EBV, Cryptococcus neoformans and JC virus, that can lead to PML, which is a fatal neurological condition characterized by subcortical white matter lesions with different neurological manifestations and without any effective treatment . NivolumThe work-up for CNS infection is based on lumbar puncture, blood cultures and brain imaging. Cerebrospinal fluid analyses should include glucose and protein concentration, polymerase chain reactions for a comprehensive panel of viral and mycobacterial nucleic acids, cryptococcal antigen, cytology and microbiological cultures. Whenever available, contrast-enhanced MRI is the preferred radiological exam, since it can highlight leptomeningeal enhancement, parenchymal inflammation and potential abscesses.It is important to rule out the cytokine-release syndrome as a differential diagnosis, since its manifestations can resemble those of CNS infection with vomiting, fever and seizures. This is a rare but severe systemic inflammatory response due to initial immune system activation after treatment with antilymphocyte antibodies , used for induction or anti-rejection therapy . These sTreatment of CNS infections includes empiric antimicrobial therapy based on ceftriaxone, ampicillin and acyclovir until the causal pathogen has been identified. A central issue with the use of antiviral drugs in kidney transplant recipients is represented by their potential nephrotoxicity. Cidofovir and, to a lesser extent, acyclovir may cause direct tubular toxicity and can also induce crystal deposition in the kidney that may result in renal failure. Hydration, slow intravenous administration and dose reduction in case of reduced glomerular filtration rate are important preventive measures. Azole antifungals are significantly metabolized by CYP3A4 and can therefore inhibit the catabolism of CsA, TAC and mTOR inhibitors, thus extreme caution is advised when using these agents in kidney transplant recipients.Immunosuppressive drugs are usually tapered in the acute phase, unless the patient’s history suggests a disproportionally high risk of graft rejection. Indeed, infections pose a challenge in balancing antimicrobial therapy and inflammatory response in transplant recipients. A reduction of the immunosuppressive regimen to contain the infection might also cause immune reconstitution inflammatory syndrome . This coGlucocorticoids are extensively employed as maintenance immunosuppression and for the treatment of acute rejection. Patients treated with glucocorticoids can suffer from different behavioral disorders, ranging from minor mood alterations and confusion to severe psychotic reactions . Dose reTransplant recipients have a well-known increased risk of malignancies compared to the general population, which is a direct consequence of reduced immuno-surveillance and increased susceptibility to infections with oncogenic viruses. The risk of developing systemic lymphoma in the first year after renal transplantation has been estimated to be 20 times higher than in the general population . Almost Clinical manifestations are variable depending on site and extension of the lesions with peripheral facial nerve palsy representing an early sign of PTLD with meningeal involvement. Definitive diagnosis usually requires a brain biopsy. Careful reduction of the immunosuppressive regimen based on the patient’s history of rejection is mandatory. Treatment options include rituximab, chemotherapy and surgery if the disease is localized.Optic neuropathy is a rare but well-recognized side effect of tacrolimus administration, which may occur from months to years after treatment initiation . PatientGuillain–Barré syndrome (GBS) is the most common acute inflammatory polyradiculoneuropathy, affecting 0.8–1.9 patients per 100,000 per year . The typCMV is the most common viral cause of GBS, and it has been associated with most cases occurring in solid organ recipients, supporting the hypothesis that in these patients viral infections can act as relevant triggers for acute nerve inflammation . GBS hasAt present, 19 cases of GBS occurring in kidney recipients have been described in the literature , 75. A sA recent single-center retrospective analysis identified 2 patients developing GBS in a cohort of 143 alemtuzumab treated kidney recipients, 92% of whom were receiving TAC-based immunosuppressive therapy (including the 2 GBS cases), within a mean interval of 6 months from alemtuzumab infusion. No recent infection was identified in either case . HoweverTaken together, these data confirm that GBS is rare in renal transplant recipients, suggesting that immunosuppression gives a certain degree of protection. This hypothesis is supported by the immunomodulatory effects of MMF, which could mitigate the immune mechanisms underlying GBS development, and the effective use of CsA in the treatment of inflammatory neuropathies . HoweverAn infectious etiology, particularly CMV re-activation, is postulated or strongly suspected in most of the cases described, which represents a higher percentage than that reported for the general adult population. Moreover, all patients receiving at least one approved immunotherapy for GBS achieved full recovery, except for one CMV-associated subject who did not fully recover lower limb strength.Therefore, the combination of antiviral medications with standard GBS therapy seems to determine the best outcome and should be offered to all patients in whom a viral etiology cannot be excluded. More information is needed to understand which is the best therapeutic approach.Myasthenia gravis (MG) is the most frequent disorder of the neuromuscular junction. It is associated with antibodies directed against the postsynaptic membrane at the muscle endplate, manifesting as fatigue and muscle weakness. An abrupt worsening of symptoms, called myasthenic crisis, may lead the patient to require respiratory support. It can occur spontaneously as part of the natural history of the disease itself, but it can also be triggered by a number of factors; infections, pregnancy, childbirth, surgery and different drugs.To date, the literature reports only anecdotal cases of myasthenic crises in patients treated with CNIs following renal transplantation , 78. HowA direct association between MG and renal transplant is therefore unlikely, but patients with an established MG diagnosis who undergo kidney transplantation must be tightly monitored. It is crucial for physicians to be aware that MG symptoms are likely to deteriorate suddenly during the perioperative period. Although there are few supporting data in the literature, conventional myasthenic crisis treatments  also seem to be effective in these patients.Tremors affect approximately one-third of renal transplant recipients, with variable degrees of symptom severity ranging from mild to highly incapacitating . CNIs arCNI-induced tremors are generally regarded as a dose-dependent effect due to over-exposure. Monitoring TAC and CsA trough levels is frequently used as a method to assess CNI exposure, but blood levels do not always correlate with intracellular concentrations and pharmacologic effect . CNI-indDose reduction of CNIs may lead to significant improvement in symptoms, and some patients may also respond to beta-blockers. Extended-release formulations of tacrolimus may provide some benefit over conventional forms, but randomized controlled trials in this context are still lacking , 87.. It has been suggested that deafness might be initiated by a sudden spike in the TAC serum level, which is later worsened by its cumulative toxic effect [Hearing loss, tinnitus or otalgia may develop in CNI-treated patients. A questionnaire administered to 521 liver transplant recipients under treatment with TAC reported that 14% developed hearing loss . Hearingc effect , accordiPeripheral toxic neuropathy is the most frequent neurological adverse event in renal transplant patients, with an overall prevalence of more than 2% . Among dPatients receiving CNI therapy show nerve function abnormalities, quantified through nerve excitability measures, consistent with nerve membrane depolarization. In 2007, the first two kidney recipients who developed sensorimotor neuropathy while on therapy with TAC were reported, showing different electrophysiological findings  of neuropathic damage . One of Several factors may contribute to the occurrence of CIDP in renal transplant patients. CNIs still play an unclear role since they have been described as provoking demyelinating neuropathy but also as having positive effects on CIDP . PatientBortezomib-induced peripheral neuropathy is a well-known adverse event of the drug. A prospective study evaluated the toxicity profile of bortezomib in 51 kidney recipients undergoing antibody-mediated rejection . The stuFinally, toxic neuropathy should be differentiated from CNI-induced pain syndrome, a rare but severe side effect. The disease should be suspected in patients complaining of movement-related, lower limb symmetric deep aching pain, especially soon after transplantation, and the diagnosis is confirmed by bone scintigraphy and magnetic resonance imaging. However, this syndrome is uncommon among kidney transplant patients, with a prevalence of 0.8–5.8% , 99. PreThe involvement of the autonomous system is common in patients with chronic kidney disease, especially due to the high prevalence of diabetes in this population. Autonomic neuropathies in these patients are frequently axonal, hence longer nerves (e.g. the vagus nerve) are affected earlier. Typical manifestations range from blood pressure disorders  due to impaired baroreceptor function, gastrointestinal symptoms  and genitourinary disorders . Some improvement in cardiovascular autonomic function has been reported in dialysis patients after renal transplantation , but othSteroid-induced myopathy can arise after a variable period of time and involves proximal muscle weakness with atrophy . These fMany neurological complications in transplant patients are related to comorbidity, hence early treatment of hypertension, diabetes mellitus and dyslipidemia is of paramount importance. Nevertheless, the best measure to prevent complications in an immunocompromised patient is to minimize immunosuppressive therapy. However, since this approach can expose transplant recipients to rejection and graft failure, knowledge of the immune status of a patient would be of great importance to decide the intensity of the anti-rejection therapy.Several studies have been conducted in the search for tests able to assess the immunological status of transplant recipients, with the ultimate goal of predicting the risk of both rejection and of complications due to over-immunosuppression . MonitorTwo additional assays were developed to characterize immunosuppression intensity, based either on the quantification of adenosine triphosphate released by CD4+ T cells , or on tMore recently, monitoring replication of torque teno virus (TTV), a non-enveloped single-strand DNA virus that is part of the human virome, has been proposed as an additional tool to assess immune function in renal transplant recipients. In a prospective observational study, low TTV loads during the first year post-transplant were associated with rejection, while high levels predicted infectious complications . An optiSince standard methods to assess the intensity of immunosuppression are still lacking, physicians should try to adjust immunosuppressive therapy on the basis of features such as age, clinical history, and comorbidities. Elderly patients experience a decline in natural and adaptive immunity , while tKidney transplant recipients are at risk of various neurological complications, mainly due to immunosuppressive drugs, and in general to their pre- and post-transplant comorbidities. These side effects can range from tremor, dizziness and paresthesia to more complex and life-threatening clinical conditions. Prevention, early diagnosis and appropriate management of these complications are critical to prevent irreversible lesions. In the past, neurological adverse events mainly occurred within a few weeks from transplant and were frequently related to opportunistic infections. In more recent years, since CNIs and other effective immunosuppressive drugs have been made available, there have been tremendous advances in the field of organ transplantation with considerable improvement in the short- and long-term survival of the patient and of the kidney allograft. Since the main focus of this review is to provide a working summary of the more common and potentially severe immunosuppression-related neurological complications in a framework based on urgency and anatomic site, our work cannot be regarded as exhaustively comprehensive, and readers interested in any of the covered topics are encouraged to pursue additional information through the cited references. More data from basic and clinical studies are needed to address the knowledge gaps in both the pathogenesis and treatment of many of these complications, and clinical decisions should take into account inter-patient variability. For these reasons, we also encourage close collaboration between nephrologists and neurologists, not only to allow correct and rapid identification of these neurological disorders, but also to provide the best therapeutic strategy in managing the delicate balance between immunosuppression and serious adverse effects.


Fungi are an integral part of the mammalian microbiota colonizing most if not all mucosal surfaces and the skin. Maintaining stable colonization on these surfaces is critical for preventing fungal dysbiosis and infection, which in some cases can lead to life threatening consequences. The epithelial barriers are protected by T cells and additional controlling immune mechanisms. Noncirculating memory T cells that reside stably in barrier tissues play an important role for host protection from commensals and recurrent pathogens due to their fast response and local activity, which provides them a strategic advantage. So far, only a few specific examples of tissue resident memory T cells (TRMs) that act against fungi have been reported. This review provides an overview of the characteristics and functional attributes of TRMs that have been established based on human and mouse studies with various microbes. It highlights what is currently known about fungi specific TRMs mediating immunosurveillance, how they have been targeted in preclinical vaccination approaches and how they can promote immunopathology, if not controlled. A better appreciation of the host protective and damaging roles of TRMs might accelerate the development of novel tissue specific preventive strategies against fungal infections and fungi-driven immunopathologies. Epithelial barriers cover the entire external and internal surfaces of our body to shield it from potentially harmful environmental influences, including toxic substances, radiation, and pathogens. Many of these barriers also fulfil essential physiological functions, such as nutrient uptake in the intestine or gas exchange in the lung. This entails challenging and partly opposing requirements for compartmentalization and permeability. One of the biggest challenges for barrier tissues is the maintenance of homeostasis with commensal microbes, while at the same time they have to prevent invasion and overgrowth of pathogenic microbes and the emergence of inflammatory disorders. Commensal microbes actively contribute to these processes, whereby the role of commensal fungi is increasingly being recognized –8. Stablin situ imaging techniques, which provide spatial information for multiparameter settings  cells and their survival and function depends on the tissue environment, all of which hampers the isolation and ex vivo analysis of these cells . Access settings  have fursettings , 20. A nthe skin .These approaches and combinations thereof have led to the discovery of various subsets of tissue-specific myeloid and lymphoid immune cells, including noncirculating memory T cells whose maintenance and function is regulated by tissue-specific environmental cues. These sessile memory T cells are referred to as tissue resident memory T cells (TRMs) . Seeding+ and CD8+ T cell subsets in mucosal tissues and the skin, but also in visceral organs, such as liver and lung. However, a T cell in any of these tissues is not automatically a TRM cell, even if it displays features of memory. TRM cells constitute a specific subset of memory T cells in non-lymphoid tissues, independent of circulating T cells. The following criteria define tissue-residency of T cells  and chemokines derived from macrophages, both of which being central to cluster formation , but that TRMs are also particularly critical for host protection during persistent infection . This inman skin  and in a of mice . In both tissues .Overall, the population of TRMs in a given tissue reflects its history of exposures to pathogenic and commensal microbes. Analysis of T cell subsets in the intestinal mucosa of children revealed that naïve recent thymic emigrants and effector memory T cells predominate during early life, whereas TRMs progressively accumulate with increasing age . The conLike all T cells, TRMs respond to TCR-mediated stimulation and activate effector functions in an antigen-specific manner. The advantage of TRMs, if compared to circulatory T cell subsets, arises from their strategic positioning within tissues, which allows immediate local recognition of infected or antigen presenting cells and a rapid recall of canonical effector functions, such as cytokine secretion or perforin- and granzyme-mediated target cell killing . TherebyUpon re-encounter with cognate antigen, TRMs can profoundly alter the local tissue environment. Through production of cytokines, they can trigger rapid adaptive and innate immune responses, including local humoral responses, activation of local innate immune cells  and recruitment of circulating lymphocytes, resulting in a remarkable cross-over between innate and adaptive immunity. Thereby, the triggering of a small number of TRMs is amplified into an organ-wide response, which can provide protection against even antigenically unrelated organisms , 51. ThiC. albicans-responsive memory T cells to explain the diverse and systemic effects that these cells can exert . CD4f flares . Attenuas in IBD . Finallys in IBD . In expe disease , 77. Sim T cells .The detrimental consequences that TRMs can have in chronic inflammatory diseases emphasize the need for tight regulation of their activity. Recent experimental evidence suggests that TRM intrinsic mechanisms control their reactivation to prevent damaging immunopathology. In a model of contact hypersensitivity to 2,4-dinitrofluorobenzene, inhibitory checkpoint receptor antagonists dramatically enhanced the magnitude and severity of eczema exacerbations . SimilarLeishmania antigen, which resulted in enhanced protection from experimental infection with Leishmania major in the skin if compared to intramuscular vaccination , which harbors both, a Dectin-2 agonistic ligand and a CD4+ T cell epitope, induced a large number of tetramer+ TRMs in the lung of mice. However, it failed to protect against lethal infection with Blastomyces .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brain metastases represent more than 50% of all cerebral tumors encountered in clinical practice. Recently, there has been increased interest in the study of extracellular vesicles, and the knowledge about exosomes is constantly expanding. Exosomes are drivers for organotropic metastatic spread, playing important roles in the brain metastatic process by increasing the permeability of the blood–brain barrier and preparing the premetastatic niche. The promising results of the latest experimental studies raise the possibility of one day using exosomes for liquid biopsies or as drug carriers, contributing to early diagnosis and improving the efficacy of chemotherapy in patients with brain metastases. In this review, we attempted to summarize the latest knowledge about the role of exosomes in the brain metastatic process and future research directions for the use of exosomes in patients suffering from brain metastatic disease. Cerebral metastatic disease is one of the most feared complications of cancer, and brain metastasis represents more than 50% of all brain tumors seen clinically. The primary source of cerebral metastatic disease is most often lung cancer followed by breast cancer. Up to 30% of cancer patients will ultimately present with cerebral metastases. Since the central nervous system lacks a classical lymphatic system, hematogenous spread is usually the dissemination mechanism .In the past decade, major advances have been made to our knowledge about extracellular vesicles. Technical difficulties in the isolation and characterization of pure populations of specific subtypes result in the co-isolation of extracellular vesicles of different subcellular origins. Therefore, the term “exosome” that is used in many articles nowadays actually refers to a mixture of small extracellular vesicles  containing both exosomal, as well as non-exosomal, components. This is why the recommendation of using the term “small EVs” instead of “exosomes” is preferable, unless the microvesicular body origin of EVs has been clearly established ,3,4,5,6.Extracellular vesicles are grouped based on their sizes, which may vary between 20 nm and 1000 nm, and based on the releasing mechanism, being represented by exosomes , microvesicles, and apoptotic bodies. Recently, interest in studying small EVs has increased. They drive organotropic metastatic spreading by preferentially accumulating at future metastatic sites ,8. OrganCancer cells have been noted for their enhanced secretion of small EVs with altered contents compared with their noncancerous counterparts. Tumor cells release significant amounts of small EVs that promote tumor growth; inhibit the immune system; and maintain a pro-metastatic phenotype by stimulating angiogenesis, invasion, and proliferation in the hosting cells. Studies have confirmed that small EVs released by primary tumor cells create a favorable environment in the secondary organs by manipulating non-immune stromal cells, mesenchymal stem cells, epithelial cells, or fibroblasts, thus preparing the premetastatic niche for the arriving tumoral cells. Additionally, exosomal release is important in driving the anterior–posterior cellular polarity used by fast-migrating cells such as human leucocytes ,16,17,18Small EVs have been isolated from human body fluids, including plasma, cerebrospinal fluid (CSF), urine, amniotic fluid, breast milk, bronchoalveolar lavage, semen, and malignant ascites . Many teExtracellular vesicles can be classified by their biogenesis mechanisms. Exosomal biogenesis is a mechanism of cellular protein quality control. Exosomes develop from the endosomal system, being formed as intraluminal vesicles within the multivesicular bodies. Invagination of the plasmatic membrane gives rise to early endosomes that, after fusing with endocytic vesicles, become recycling endosomes and late endosomes. Inward budding of the endosomal membrane results in the accumulation of intraluminal vesicles within the late endosomes. These late endosomes containing intraluminal vesicles are termed “multivesicular bodies” and may fuse with lysosomes or with the cellular membrane. If the multivesicular bodies fuse with lysosomes, their content is degraded, but if they fuse with the cellular membrane, they release intraluminal vesicles into the extracellular space as exosomes—Exosomal biogenesis is catalyzed by many factors, including tetraspanins such as CD9 and CD63 that accumulate in the endosomal membrane, reorganizing it. These tetraspanins can be used as “specific” exosomal biomarkers along with CD81, another tetraspanin specific to exosomal biogenesis. The endosomal sorting complexes required for transport (ESCRT) play an important role in the formation of intraluminal vesicles. Four different ESCRT types—O, I, II, and III—have been identified so far . Exosoma3 as a consequence of insufficient blood supply caused by aberrant tumoral microcirculation [Hypoxia is responsible for tumoral progression, cancer metastasis, and therapy resistance and is present in all solid tumors with volumes over 1 cmculation . For solculation ,28. In hculation .Tumors are made of many genetically and phenotypically different cell subclones. It seems that, within the primary tumor microenvironment, different subclones can transfer oncogenic features into neighboring cells using small EVs . For exaSmall EVs released by the primary tumor cells contain molecular constituents that induce cellular immune dysfunction, reducing the antitumoral immune response either by altering the function of immune cells or by modifying the nonimmune stromal cell phenotype . T-cell The concept of the premetastatic niche was first proposed and demonstrated by David Lyden . A premeIn the clinical stage of cerebral metastases, small EVs released from secondary sites seem to be as important as those released by the primary cancer cells; for example, the small EVs released by normal astrocytes decrease the expression of the phosphatase and tensin homolog gene (PTEN), which is an important tumor suppressor in brain metastases. Zhang et al.  showed tThe metastatic process, also known as the metastatic cascade, is composed of the following steps that tumor cells must undergo to successfully disseminate into secondary organs: local invasion, immune response survival, intravasation inside the bloodstream, and extravasation in the secondary organs. Small EVs enhance systemic invasion and cancer cell progression in the metastatic cascade . For specific brain metastases to develop, tumor cells must pass through the tight junctions of the blood–brain barrier (BBB) and survive inside the brain parenchyma. The majority of cancer cells die instantly after crossing the BBB, so, even now, the underlying mechanisms of the cerebral metastatic process remain to be clarified . DespiteIn the metastasis of lung cancer cells towards the brain parenchyma, the BBB permeability markedly increases, allowing circulating cancer cells to penetrate the brain parenchyma; however, these mechanisms are not fully understood. Wu et al.  showed, Tominaga et al.  showed tWhen discussing the dissemination mechanism of cancerous cells that must traverse the BBB, we may wonder about the mechanism of adhesion of the tumoral cells to the endothelial cell layer of the brain, which may be a crucial step that precedes the transmigration of cancerous cells. Fazakas et al.  used atoSmall EVs modulate vascular permeability and stimulate neoangiogenesis, two steps that are crucial in premetastatic niche establishment, resulting in the initial extravasation of tumor cells followed by metastatic cancer cell development in secondary organs. For example, MDA-MB-231 breast cancer metastatic cells release small EVs containing miR105 that help destroy the endothelial cell barrier by downregulating ZO-1 (zonula-occludens 1 protein) tight junctions, increasing the permeability of the endothelial barrier and permitting cancer cell metastasis into secondary organs ,56. Lu eMicroglia are the immune cells found inside the brain that are activated by many pathological situations, such as infection or cancer of the neuroaxis. Microglia can have both tumor-suppressive (M1) and tumor-promoting (M2) roles. It has been shown that, preferentially inside the brain, XIST (X-inactive-specific transcript) works as a metastatic suppressor gene, as demonstrated by XIST downregulation promoting epithelial-mesenchymal transition (EMT), MSN/c-Met, and small EV miR-503 release, all of which leads to the development of metastatic traits in tumor stem cells and microenvironment reprogramming via small EV-mediated communication. The increased secretion of small EV miR-503 results in reprogramming of the microglia from M1 to M2, leading to immune suppression and the release of growth factors that promote tumor cell survival and migration in patients with breast cancer, thus promoting brain metastasis. Serum small EV miR-503 may be used in the future as a biomarker to assess the risk of developing brain metastases in patients with breast cancer .Exosomes have various transmembrane, intraluminal, or surface-attached proteins. Tetraspanins are integral exosomal membrane proteins that mediate the exosomal inclusion of various proteins, such as integrins, which play a crucial role in the development of the premetastatic niche and organotropism of lung and liver metastases. Curiously, small EVs released by brain metastases pack very small amounts of integrins, suggesting that other exosomal molecules may support brain metastasis ,59. HoweOnly a few organs host deposits of metastatic cells, although cancerous cells can be found in the vascular beds of many organs, further emphasizing the role of small EVs as key factors in the metastatic process. Rahman et al.  showed tAnother protein carried by small EVs and released from brain metastatic lung and breast cancer cells is the cell migration-inducing and hyaluronan-binding protein (CEMIP or KIAA1199). This protein promotes brain metastasis by inducing a proinflammatory vascular niche in the early stages of metastatic colonization. Studies show that high CEMIP expression in primary lung or breast cancer cells is associated with shorter intervals between primary cancer and the metastatic stage, the rapid evolution of brain metastases, and a poor prognosis for the patient. CEMIP expression is very high in small EVs released by lung and breast brain metastases, although not in metastases located elsewhere or even in the primary lung or breast cancer cells. Additionally, CEMIP seems to be selectively packed in exosomes, because it occurs in ten times larger quantities in small EVs compared with brain metastatic cells . S100A16, a protein in the S100 family, increases in SCLC cells located in the brain microvascular microenvironment. Small EVs derived from human brain microvascular endothelial cells (HBMEC) control S100A16 elevation. Experiments have shown that SCLC cells treated with small EVs released from HBMEC increase the S100A16 level, resulting in increased apoptosis resistance under stress and increased cell proliferation. Additionally, S100A16 upregulates PHB-1, a protein located in the inner mitochondrial membrane, preserving the mitochondrial membrane potential and, thus, improving the survival of metastatic SCLC cells in the cerebrum . Puigdelloses et al.  showed tThe small EVs released from metastatic breast cancer cells increase the expression of miR210, annexin II, and integrinβ3. Small EVs released by metastatic lung cancer cells present an increased expression of TGF beta and IL-10. The drawback when using small EVs as biomarkers is that all the cells release small EVs that can interfere with the small EVs released by tumoral cells. One way to solve this situation is by using cancer-specific markers to separate tumor-secreted small EVs from the small EVs released by normal cells [Small EVs may be used as a minimally invasive way to track brain metastases or other primary brain tumors and assess the response to various treatment methods, such as surgical treatment and chemo- or radiotherapy, by obtaining exosomal profiles before and after treatment. The use of small EVs as liquid biopsies could be beneficial for situations in which imaging findings do not reflect the present tumor biology, as when a differential diagnosis must be made between tumor recurrence and radiation necrosis or tumor pseudo-progression, which results in an increased contrast enhancement on imaging. Liquid biopsies may also be used when a patient is treated with antiangiogenic agents that reduce the contrast enhancement by decreasing the permeability of the tumor vascularity without actually reducing the tumoral size. Using small EVs as liquid biopsies will significantly decrease the risks associated with classic biopsies ,63. Chemotherapy is known to be inefficient in brain metastases, because drugs have to traverse the BBB to be efficient. In the future, small EVs will be used as carriers for therapeutic agents across the BBB. Liu et al.  used CXCBetter understanding the process of tumor exosomal biogenesis may lead to decreased metastasis through the development of new techniques to control tumor exosomal production. Goals for the future include the development of new ways to identify the noncancerous premetastatic sites developed in secondary tumor-free organs early by combining the advances in serum biomarker identification, imaging, and the continuously accumulating knowledge about the premetastatic niche development. The early identification of premetastatic niches could allow them to serve as prognosis biomarkers and therapeutic targets to prevent metastasis development ,68.Recently, cancer stem cell (CSC) studying has gained much interest. It is known that CSCs contribute to tumorigenesis, metastasis, and therapeutical resistance, because these cells have the ability to self-renew and to differentiate into various cell types. CSC present chemo and radio resistance, attributable to EMT, DNA damage checkpoint activation, the upregulation of CSC markers, and the signaling pathways. This makes them extremely sought-after targets in the battle against resistance to treatment and tumoral recurrences. CSCs have been identified in solid tumors such as brain gliomas, breast, ovarian, colorectal, prostate, and pancreatic cancer, as well as in hematologic malignant diseases such as leukemia. Small EVs released by the CSC represent a new area of interest, because they transmit information between CSC and non-CSC, resulting in the activation of the CSC and modification of the surrounding microenvironment, finally contributing to cancer progression. Brain tumor CSCs express the exosomal markers TSG 101 (tumor susceptibility gene) and flotillin 1 which can suppress T-cell activity by the upregulation of tenascin-C. New ways of targeting these small EVs may finally lead to elimination of this CSC, resulting in curing the patient. This subject still represents a novelty, and further studying is needed ,70,71.Small EVs represent an important target that may be used for early cancer diagnosis, prognosis prediction, cancer treatment, or to track the responses to various treatment methods. Their use in the diagnosis of central nervous system metastasis as liquid biopsies derived from blood or CSF shows promise, especially in deep-seated lesions where stereotactic biopsies may be dangerous or in differential diagnosis with primary gliomas. Small EVs can be used not only for diagnostic purposes but also for molecular tumor profiling.