The mammalian gut contains a large, complex community of microorganisms collectively termed the microbiota. It is increasingly appreciated that gut microbes are closely integrated into mammalian physiology, participating in metabolic symbiosis, promoting immune function and signaling to a wide variety of distant cells, including the brain, via circulating metabolites. Recent advances indicate that microglia, the brain’s resident immune cells, are influenced by microbial metabolites at all stages of life, under both physiological and pathological conditions. The pathways by which microbiota regulate microglial function are therefore of interest for investigating links between neurological disorders and gut microbiome changes. In this review, we discuss the effects and mechanisms of microbiota-microglia signaling in steady state, as well as evidence for the involvement of this signaling axis in CNS pathologies. Some such interactions are mutualistic, such as co-operative food digestion and nutrient synthesis, though some aspects of physiology are influenced by the mere presence of commensals or by infection by pathogenic species. Thus, the physiology of most animals can be best understood as a meta-organism comprising the host and a huge diversity of commensal microorganisms collectively referred to as the microbiota. Host-microbiota interactions are increasingly understood to be fundamental in shaping diverse aspects of mammalian biology, including development, metabolism, immunity and neurological function. The link between intestinal microbiota and brain function has been extensively described, and is commonly referred to as the microbiota-gut-brain axis.2 Although separated from commensal microbes by both the gut barrier and blood–brain barrier, the presence of intestinal microbes has been demonstrated to regulate microglial phenotype and functions, both at steady state and under disease conditions. Given the increasing interest in the gut microbiome as a regulator of neurological disorders, direct communication between intestinal microbiota and microglia is an intriguing concept which may prove to be a central mechanism linking the microbiome to various brain diseases. In this review, we explore recent advances in understanding the mechanistic links between gut flora and microglia, as well as their consequences for neurological disorders.Microglia are one of the four primary cell types comprising the mammalian central nervous system (CNS). As the brain’s primary immune population, they contribute not only to the development and maintenance of the CNS, but also critically regulate CNS disease states such as neurodegeneration, autoimmunity and neurodevelopmental disorders.+ macrophages in the embryonic yolk sac, which proceed to colonize the embryonic brain at around E9.5, quickly spreading to all developing brain structures.3–6 Due to the establishment of the blood–brain barrier at E13.5, the brain is shielded from subsequent waves of hematopoiesis originating in the fetal liver and bone marrow. Thus, there is no replacement of yolk-sac-derived microglia with monocyte-derived cells under homeostatic conditions,7 as is observed in other tissues.8 Instead, microglia self-renew throughout life via tightly regulated local proliferation and apoptosis.10 After first colonizing the brain at E9.5, microglia undergo several stepwise maturation stages , scavenger receptors and triggering receptor expressed on myeloid cells 2 (TREM2). Moreover, mature microglia express the receptors P2RY12 and P2RY13, conferring exquisite sensitivity to purinergic stimuli. Secondly, their long, elaborately branched processes are remarkably motile, undergoing extremely dynamic, rapid rearrangements and making contact with surrounding CNS cells on a constant basis.32 Thus, via their extensive sensome and unceasing motility, microglia scan the entire CNS space once every few hours, and are the primary responders to any disturbance in CNS homeostasis.Adult microglia exhibit several marked adaptations to their role as the primary immunocompetent cells of the brain. Firstly, their unique transcriptomic signature encompasses a large repertoire of genes involved in immunosurveillance.P2ry12, Tmem119 and Mafb, and induce genes involved in effector functions, many of which vary depending on the specific insult. Recent studies support the existence of multiple states of reactive microglia which can be induced by different pathologies such as neurodegeneration and autoimmunity and engage diverse reactive functions including phagocytosis, proliferation, antigen presentation and inflammatory cytokine secretion.33–35 Whilst microglial responses are undoubtedly critical for effective resolution of insults and preventing damage to the CNS, there is ample evidence to suggest that inappropriate microglial activity can be a causative factor in various CNS diseases.35 Microglia are therefore a key candidate for transducing environmental factors such as microbiome-related signaling to the exacerbation or amelioration of CNS pathology.Following CNS insults such as injury, infection and neurodegeneration, microglia sense local cues and undergo transformation to reactive phenotypes. In general, these reactive microglia downregulate some homeostatic markers such as 36 In this sense, the gut microbiome is often described as an additional metabolic organ, assisting with fiber fermentation, tryptophan metabolism and detoxification of xenobiotics, amongst a wide range of other pathways. However, the effect of bacterial metabolism on mammalian physiology goes far beyond simple nutrient provision. Hundreds of bioactive metabolites are produced by the microbiota, circulating throughout every organ and affecting the activity of huge numbers of target cells.37–39The gut microbiota are known to exert extensive effects on host physiology via a variety of mechanisms. The most prominent mutualistic function of gut bacteria is metabolism – collectively the bacteria inhabiting the gastrointestinal tract contain many more metabolic genes than humans possess, thus allowing much greater metabolic flexibility than would otherwise be possible.1 The microbiota are critical players in the gut-brain axis, and can affect CNS physiology by directly modulating neurotransmission and neurodevelopment, by signaling through the vagus nerve, as well as by modifying endogenous metabolism, endocrine signaling and immune system activity  or could involve other cell types, or indeed could be secondary to effects on the Purkinje neurons themselves. Nonetheless, this study suggests microglia could be critical in linking early-life neuroimmune interactions and neurodevelopment to microbiome composition. Further studies should seek to determine how early gut microbiota influence microglia, and whether this communication axis could affect the genesis of neurodevelopmental disorders.Interactions between microglia and colonizing commensals are also important for early postnatal neurodevelopment.44 Intriguingly, the transcriptomic phenotype of germ-free microglia appears to be immature compared to SPF controls, with increased expression of proliferative markers and decreased expression of genes relating to immune function  and other markers of mature adult microglia .43 It is therefore increasingly appreciated that colonization with gut microbes may provide an environmental cue driving microglia to assume their fully mature phenotype, which occurs naturally around the same time. Moreover, since ablation of the microbiome in SPF animals via broad-spectrum antibiotics largely phenocopies the effect of germ-free housing on microglial phenotype, this instruction clearly occurs constitutively, rendering microglia sensitive to real-time perturbations in gut microbe composition.43 Interestingly, colonization of germ-free animals with a defined repertoire of 15 species known as altered Schaedler flora (ASF) was insufficient to reproduce the effect of SPF housing.43 This suggests that complex microbiota may be better able to support microglial maturity than other, defined communities. However, it is unclear whether the inability of ASF flora to replicate the effects of SPF housing is truly related to complexity, or rather to insufficient production of particular metabolites such as SCFAs, which might be adequately provided by different species in a similarly defined consortium. Remarkably, it was recently reported that, of the 3 major SCFAs produced by the gut biota , only acetate is in fact necessary and sufficient for microglial maturity, with neither propionate nor butyrate able to influence the phenotype of germ-free microglia.44 Thus, production of acetate alone may be one of the most critical parameters when considering the effects of microbial communities on microglial state.In adult animals, gut bacteria exert a constitutive influence on microglial physiology via release of short-chain fatty acids (SCFA) . Microgl43 Similarly, infection with lymphocytic choriomeningitis virus (LCMV) in germ-free animals produces lower induction of Il1b and Tnfa, as well as the immediate-early genes Fosb, c-fos and c-jun compared to SPF controls.43 Interestingly however, germ-free microglia displayed normal induction of the antiviral signaling molecules Cxcl10, Irf7 and Isg15 following LCMV infection.43 This could indicate that lack of microbiota induces a specific signaling defect in microglia which impairs certain response pathways but leaves typical antiviral signaling mediated by interferons and RNA sensing pathways intact. The exact nature and mechanism of the signaling alterations induced by lack of microbiota are still unclarified, but could prove critical when drawing mechanistic links between microbiota perturbations and microglial disease responses.The immature phenotype of germ-free microglia is not limited to effects on morphology and gene expression in homeostasis. Instead, germ-free microglia also exhibit defective responses to immune stimuli. In SPF mice, intracerebroventricular (i.c) injection of the bacterial endotoxin lipopolysaccharide (LPS) induces a robust TLR4- and NF-κB-dependent response resulting in upregulation of inflammatory mediators including TNF-α, IL-1ß and CCL2. In germ-free animals, this response is markedly diminished, indicating an immunologically immature state.44Given the separation between microglia and commensal microbes, interactions are generally only possible via circulatory factors or via altered activity in an intermediate cellfor example, neuronal signaling via the vagus nerve. On the simplest possible level, molecules produced by gut microbes can enter the blood through the intestinal wall, subsequently entering the brain and acting on microglia directly to alter their physiology . However44 Thus, while SCFAs had generally been regarded as a single class of metabolite, it appears that their effects on microglia are divergent, with acetate being the primary mediator of microglia-microbiota signaling.44 The mechanism by which acetate affects microglial phenotype remains largely unclarified, though several potential pathways have been identified.Currently the best-studied microglia-microbe interactions occur via circulating SCFAs, which are produced by gut bacteria via fiber fermentation. The three most abundant SCFAs produced by the microbiota are acetate, propionate and butyrate, consisting of 2-, 3- and 4-carbon chains, respectively. It was recently discovered that acetate alone is able to reverse many features of the germ-free microglial phenotype, while propionate and butyrate are entirely ineffective.44 This same report also linked acetate availability to metabolic alterations in germ-free microglia. Specifically, germ-free microglia exhibit increased mitochondrial mass compared to SPF controls, which is coupled to reduced activity of the mitochondrial electron transport chain complex II.44 This defect is rescuable by acetate supplementation and may relate to lack of acetyl Co-A, resulting in increased generation of oxaloacetic acid, a known complex II inhibitor.44 Whilst it is unclear whether this metabolic defect is related to the other functional alterations in germ-free microglia, it is feasible given the now widely recognized role of metabolism as a central regulator of macrophage function.46 These data therefore support a model whereby microbiota modulate microglia by providing a carbon source required for normal metabolism, thus forming a direct link between diet, gut microbiota composition and microglial state. This idea is particularly compelling given that the primary receptor for acetate, FFAR2,47 is not expressed in the brain, thus making direct effects of acetate on microglia via GPCR signaling unlikely.43Recent data using radiolabelled carbon tracing indicate that gut-derived acetate in the blood is capable of entering the brain and is incorporated into the microglial Krebs cycle.48 Exposure to Aβ is well described to induce a shift in microglial metabolism toward glycolysis, which over time supports inflammatory polarization and subsequent inhibition of phagocytosis, thus promoting accumulation of amyloid plaques.49–51 Indeed, GF microglia exhibit lower induction of glycolytic and proinflammatory genes in the 5xFAD model than their SPF counterparts, which is largely dependent on acetate.44 Thus, acetate availability appears to constitutively modulate both dependence on glycolysis and inflammation in amyloid-exposed microglia, which may result in phagocytic suppression and increased plaque accumulation. However, exactly why this occurs remains unclarified.It is especially interesting to consider the effects of acetate on microglial metabolism in the 5xFAD AD model, where germ-free animals exhibit greater microglial phagocytosis and reduced plaque pathology, which is reversible by acetate supplementation.43 However, supplementation of germ-free animals with propionate, another FFAR2 agonist, does not affect microglial phenotype, with acetate being the only SCFA capable of reproducing microbiota-induced microglial maturation. As such, it is possible that FFAR2 is dispensable for the effect of acetate on microglia, but its deletion phenocopies acetate deprivation via a separate, unclarified mechanism.Additional explanations for the maturation-inducing effect of acetate on microglia include protein acetylation and activation of GPCR signaling pathways. Indeed, microglia in mice lacking the SCFA receptor FFAR2 exhibit similar defects to those from germ-free animals despite a lack of FFAR2 expression in the brain, indicating that microglia could be affected by SCFA signaling in other organs.52 For example, the transcription factor NF-κB, which is critical for the generation of inflammatory responses, relies on acetylation of the p50 and p65 subunits for DNA binding and full transcriptional activity, respectively.54 LPS elicits classical NFκB-dependent responses which are blunted severely in germ-free microglia. This raises the possibility that the dimished LPS responsivity observed in the absence of microbiota could be due to lower acetate availability leading to insufficient acetylation of NF-κB or other immunologically important proteins. However, to date no studies have examined the effect of microbial acetate on acetylation of the general microglial proteome.Protein acetylation constitutes another route by which acetate availability might directly influence microglial physiology. Acetylation is an extremely common post-translational modification with profound consequences for the function of various immunological proteins.55 and are a key mechanism regulating immune cell effector functions following stimulation.57 Acetate supplementation produces increased histone acetylation in various organs,58 including the brain,59 and microbiota have been reported to regulate patterns of acetylation across a variety of histone sites via release of SCFAs.58 Gut microbiota-derived acetate may affect histone acetylation by increasing the availability of acetyl-CoA, a necessary substrate for HAT activity, or via inhibition of histone deacetylases (HDACs).59 In microglia, gut microbiota have been shown to regulate acetylation at the 9th lysine of histone 3 (H3K9),44 a modification associated with increased promoter activity. Whilst levels of H3K9 acetylation appear to decrease overall under GF conditions, many genes also display increased H3K9 acetylation compared to SPF microglia.44 This is in agreement with a previous report which identified differentially accessible chromatin regions between GF and SPF microglia via ATAC-Seq beginning in early development.12 Thus, the microbiota clearly regulate microglial histone acetylation, which could underly some aspects of their effect on microglial function. However, whether or not this effect can be attributed to acetate is unclear, since propionate and butyrate are also known HDAC inhibitors capable of influencing epigenetic state60–62 and the effect of individual SCFAs on microglial histone modification has not been assessed. Moreover, it is unknown whether microglial histone modification patterns are controlled dynamically by the microbiota or are imprinted during development and remain static thereafter, as was recently suggested for the liver.63 Finally, it would be helpful to dissect precisely which aspects of the germ-free microglial phenotype are dependent on epigenetic modifications, and whether this could account for the differing effects of antibiotic treatment in SPF animals versus acetate supplementation in GF conditions.48Another facet to protein acetylation involves epigenetic modification of histone proteins by histone acetyltransferases (HATs). Acetylated histones generally result in more accessible chromatin and increased gene transcription,44 This could be explained by lingering epigenetic differences between GF and SPF animals, and/or could imply the presence of extra signaling factors which regulate microglial phenotype more subtly. Aryl hydrocarbon receptor (AhR) ligands have been demonstrated to influence microglial state during CNS autoimmunity,64 but their effects under homeostatic conditions have not been reported. Microglia are also affected by microbial products during embryonic and postnatal development, but the molecular players are entirely unknown. Acetate has been shown to be differentially abundant in SPF versus germ-free embryos, along with a variety of compounds including the AhR ligand indoxyl-3-sulfate, trimethylamine-N-oxide (TMAO), hippurate, N, N, N-trimethyl-5-aminovalerate (TMAV) and imidazole propionate.65–67 One recent study identified defective thalamocortical axon development in germ-free embryos, which is rescued by maternal supplementation with the latter 4 compounds listed above, but not by SCFAs including acetate. Whether embryonic and early postnatal microglia could also be regulated by these compounds, acetate, or AhR ligands would provide an interesting basis for future studies.Taken together, the evidence for a direct microbiota-microglia communication axis mediated by acetate is compelling, but key questions remain around the precise nature of the effect, which may involve multiple parallel functions of acetate as both a signaling molecule and metabolic substrate. A major question moving forward is what role other microbial metabolites might play in instructing microglial phenotype. Acetate supplementation reverses most of the metabolic and phenotypic differences between GF and SPF microglia, but does not completely normalize the transcriptome under either homeostatic or pathological conditions.2 Signaling from the microbiota to microglia has therefore been suggested as an environmental factor capable of promoting or ameliorating disease. In support of this idea, microbiota-dependent influences on microglial states have been reported to exhibit a high degree of sex-dependence,12 which could help account for the marked differences in susceptibility to certain CNS diseases observed between men and women. A vast quantity of literature has in recent years been generated suggesting roles for microbiome dysbiosis in the pathology of numerous neurological conditions. Since this is too extensive to review in full, we focus here on critically reviewing the most compelling evidence linking communication between microbiota and microglia to neurological disorders.Microglia are active regulators of a variety of CNS pathological states, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, as well as neuroinflammatory and neuropsychiatric conditions.68 Amyloid plaques are composed of high-order aggregates of amyloid-beta (Aβ), a peptide produced by cleavage of amyloid precursor protein (APP).69 While the pathology of AD is complex and incompletely understood, excessive accumulation of Aβ is thought to be a major trigger of subsequent disease processes such as tau hyperphosphorylation, synaptic dysfunction and neuronal loss.69–71Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by progressive brain atrophy and profound memory loss, which is accompanied by deposition of amyloid plaques and overabundance of hyperphosphorylated tau protein.73 More specific differences on the level of individual phyla and genera have been reported, but in some cases are inconsistent between different studies72–77 (reviewed by78). For instance, Vogt et al.72 noted decreased abundance of Actinobacteria in AD patients, whereas Ling et al.73 observed an increase. Similarly, increased abundance of Bacteroides is noted by some, but not other studies.77 One relatively consistent point, however, is that the abundance of Firmicutes appears to be generally decreased in AD patients.72–77 Additionally, some functional relationships have been proposed between AD pathology and the balance of inflammatory versus anti-inflammatory species such as Eschieria/Shigella, E. rectalis and F. prauznitii.75 While it is entirely feasible that changes in gut microbiota aggravate or promote AD, and evidence exists for altered microbiome composition in AD patients, human clinical studies have thus far been entirely correlative. Moreover, since AD-associated pathological changes begin decades prior to the onset of clinical symptoms,70 the most relevant changes in gut biota may be missed by studies focusing on patients with diagnosed AD, and future studies tracking temporal changes in microbiome composition prior to AD onset may be more informative.The microbiota are proposed to play a major role in AD, which could occur at least partially via microglia-mediated mechanisms. Several studies have reported decreased richness and diversity of fecal microbiota in AD patients compared with healthy controls.79 Subsequent studies have validated that germ-free housing or antibiotic treatment reduce plaque pathology in the APPSwe/PS1ΔE9,81 APPPS1-2181–83 and 5xFAD48 models. Mechanistically, this effect seems to be independent of amyloid processing pathways,48 and instead reflects altered microglial activity.48 Microglia from germ-free animals exhibit greater induction of phagocytic genes such as Axl, Apoe and Clec7a, as well as higher expression of reactivity markers such as Cst7 compared to SPF controls.84 This is coupled to increased levels of amyloid phagocytosis in germ-free microglia, indicating that microbiota interactions direct critical microglial functions in AD models.48 Interestingly, recent studies report that, similarly to the steady-state phenotypic differences observed in germ-free mice, the effect of microbiota on amyloid pathology appears to be mediated primarily SCFAs, specifically acetate.84 Thus, clear evidence exists for an acetate-mediated microbiota-microglia signaling axis which promotes amyloid accumulation in AD mouse models. Most intriguingly, a recent study in human AD patients reported that amyloid burden correlates positively with serum acetate levels, but negatively with serum butyrate and propionate.85 This supports a model whereby acetate may act via microglia to promote amyloid pathology in AD, whereas butyrate and propionate are unable to modulate microglial physiology and instead are protective due to effects on other cell types. While causative associations between individual microbes or metabolites and human AD remain unproven, these results suggest that AD interventions targeting gut-derived SCFAs should pay close attention to the role of acetate versus propionate and butyrate in modulating amyloid burden.Whilst mechanistic associations between gut microbiota variation and AD pathology are difficult to obtain in humans, there is now a wealth of corroborating data confirming that mouse models of AD are extremely sensitive to changes in gut bacteria. The first study to address this issue demonstrated that antibiotic administration to APP/PS1 mice (high-dose for one week followed by chronic low-dose) reduced amyloid plaque burden.86 Symptoms include progressive motor dysfunction along with cognitive impairment, depression, and sleep disruption.Parkinson’s disease (PD) is a complex neuropathology involving degeneration of dopaminergic neurons in various brain regions such as the substantia nigra, along with extensive intracellular accumulation of aggregated α-synuclein protein.87 and that gut inflammation and motility problems are common in PD patients. It has even been postulated that α-synuclein aggregates may in some cases originate in the gut and propagate to the brain in a prion-like manner via the vagus nerve, though this idea remains controversial.87–89 A role for the microbiota in regulating PD pathology could therefore be multifaceted, influencing the local generation of toxic α-synuclein species or distantly affecting microglial phenotypes in affected regions of the CNS, as occurs in AD models. Some studies have demonstrated altered gut microbiota composition in PD patients, though the results are likely affected by drug treatment and, as with AD, it is unclear whether gut flora changes precede brain pathology.90Gut-brain signaling has long been suggested to be of particular relevance for PD, especially considering that α-synuclein pathology is often observed in the gut,91 Similarly to in AD models, the detrimental effects of complex gut biota on neuropathology in ASO mice are reproducible by supplementation with SCFAs.91 Interestingly, colonization of GF ASO mice with microbiota samples from PD patients generally (but not invariably) produced greater motor impairment than with samples from healthy controls.91 However, it is unknown whether this effect is due to differences in SCFA production between PD-derived and control microbiota or whether other mechanisms are responsible.To date, the only direct evidence for involvement of the gut microbiota in PD comes from rodent studies. In an α-synuclein overexpression (ASO) model, GF mice were protected against gut motility defects, microglial reactivity, brain inflammation and ultimately motor deficits.92–95 However, it has also been suggested that, due to the frequent alterations in gut function accompanying PD, stool SCFA measurements may not accurately represent systemic exposure compared to healthy controls.95 Measurements of circulating SCFAs in serum are less common, but have been variously reported as decreased,96 increased,95 or unchanged97 in PD patients. Moreover, correlations between individual SCFAs and clinical symptoms are most often reported for propionate and butyrate,97 which in mice do not directly affect microglia.44 Thus, there is currently little evidence to support acetate-mediated microbiota-microglia signaling as a causative mechanism in PD patients. While this does not rule out a role for the microbiota in modulating microglial phenotype in human PD, it may be that other metabolites are responsible,98 or indeed a more complex mechanism such as via systemic immunity, gut barrier integrity or neuronal signaling. Further mechanistic studies in mouse models may prove useful to elucidate exactly why PD patient-derived microbiota elicit greater neurodegeneration in mouse models, and whether microglia are critically involved.Most studies assessing SCFA levels in PD patients have found decreased concentrations in fecal samples, suggesting dampened SCFA production.99 While the disease course of MS is highly variable, most patients eventually experience irreversible neurological damage, impairment and disability which continues to worsen over time. The causes of MS are unknown, though previous infection with Epstein-Barr virus (EBV) appears near-universal amongst MS patients, and may promote the generation of cross-reactive T cells via molecular mimicry.100 As a primarily immune-mediated condition, immunomodulatory therapies in MS have shown some clinical success in reducing relapse frequency and slowing disease course.99Multiple Sclerosis (MS) is an autoimmune condition generally caused by autoreactive immune responses directed against myelin components, resulting in the formation of brain lesions exhibiting prominent inflammation, demyelination and blood–brain barrier disruption.101 One the one hand, microglia can contribute to the intense inflammation typical of MS lesions, inducing neuronal damage and demyelination.101 On the other hand, microglia are also critical for phagocytosis of myelin debris and stimulating oligodendrocyte differentiation and remyelination.101 Thus, microglia constitute a key target cell for any interventions aimed at modifying the course of MS. Unlike typical neurodegenerative conditions, the peripheral immune system is heavily involved in MS, and overall pathology is influenced by the properties of both CNS-resident cells and infiltrating peripheral cells. Thus, whilst there is evidence that gut bacteria are capable of modifying MS pathology, it must be considered that their effects can be mediated not only through changes in microglia but also in peripheral immunity. Indeed, a variety of studies have demonstrated that gut flora are required for the generation of autoreactive T-cell responses in experimental autoimmune encephalomyelitis (EAE), a commonly used model of MS.103 Thus, germ-free animals are generally resistant to EAE pathology due to defective T-cell differentiation. In humans, differences in the ability of gut bacteria to stimulate inflammatory versus regulatory T cell responses may constitute a key environmental factor determining MS risk.104 It is also for this reason that GF animals are of limited use for studying microbiota-microglia signaling in EAE, and therefore only few studies have successfully demonstrated mechanistic links between gut biota and microglial phenotypes in EAE models.Multiple states of reactive microglia can be observed in MS, and likely play multifaceted roles in regulating the progression of pathology.64 Mice harboring AhR deletion in microglia following transient CX3CR1-CreERT2-mediated recombination exhibit exacerbated clinical scores after immunization with MOG peptide.64 This was found to be due to reduced AhR-dependent secretion of TGF-α and increased production of VEGF-B from microglia, causing increased pro-inflammatory astrocyte polarization. AhR ligands are produced by the gut microbiota via tryptophan metabolism, and restriction of dietary tryptophan in WT mice phenocopied the effect of microglial AhR deletion.64 Conversely, supplementing a tryptophan-deficient diet with AhR ligands such as indole-3-carbinole (I3C) rescued the worsened EAE pathology in WT, but not microglial AhR-deficient mice.64 Thus, gut flora are capable of influencing microglial phenotypes in EAE independently of changes in T cell-mediated immunity, and may be critical in transducing diet-related environmental variables to EAE risk. These results build on a previous study indicating that AhR ligands can also signal via astrocytes, and that AhR signaling may form part of the protective influence of interferon-beta in MS.105 Thus, microbial tryptophan metabolism and AhR ligands are a promising lead for harnessing endogenous microglia-microbial signaling pathways in MS, and efforts to validate their role in human subjects are ongoing.106One key study examining microglia-microbiota crosstalk in EAE did so by focusing on the aryl hydrocarbon receptor (AhR), a major mediator of gut-immune signaling.107 The etiology of ASD is poorly understood, but is generally thought to be a multi-hit process involving genetic predisposition in combination with environmental triggers during critical developmental periods.108 Gastrointestinal symptoms are common in individuals with ASD, suggesting a link between gut function and ASD symptoms.109 The gut microbiota is a prime example of an environmental factor which is established during early development and proceeds to regulate both brain and gut physiology, and is often dysregulated in ASD.1 There is currently intense interest in microbiota-based therapies for ASD, some of which have already shown promising results in initial trials.110Autism Spectrum Disorder (ASD) comprises a group of related neurodevelopmental disorders whose manifestations vary widely in both type and severity but can include learning and social impairment as well as repetitive behaviors.111 Indeed, some degree of microglial abnormalities and brain pathology have been noted in ASD patients.112–116 However, altered microglial states could occur downstream of neuronal abnormalities in ASD and definitive causal associations are still lacking.Microglia are presumed to be involved in ASD pathology due to their intimate contribution to neurodevelopment and ability to regulate brain function through release of inflammatory mediators and neurotrophic molecules.118 and offspring of mice injected with the viral mimetic poly(I:C) during pregnancy exhibit a phenotype which shares some features with ASD, including social interaction deficits and increased repetitive-like behaviors.119 This effect depends on induction of an acute inflammatory response in the maternal serum, including IL-6 and IL-17a.120–122 Maternal cytokine elevation results in subsequent production of inflammatory mediators in the fetal brain,123 and increased concentrations of some cytokines can be detected during postnatal development,124 though precisely which mediators are altered at which time points vary between studies.125 Microglia from MIA offspring display an altered phenotype characterized by premature induction of the mature gene signature in neonatal pups, as well as decreased phagocytosis and higher basal expression of inflammatory genes in adulthood.126 While these results in combination with human data suggest that microglial dysfunction can promote ASD pathogenesis, the exact contribution of microglia to cognitive and behavioral outcomes is still unclear, and many studies report conflicting results with regard to microglial reactivity following MIA.127Currently some of the best evidence for causative roles of microglia and microbiota in ASD come from models of maternal immune activation (MIA). Viral and bacterial infections during pregnancy are a known risk factor for the development of ASD in humans,122 Thus, the ability of viral infection to promote ASD may depend on the presence of commensals which direct maternal immune responses. Gut bacteria may also have constitutive effects on ASD pathology, since colonization of GF mice with gut biota derived from individuals with ASD promoted behavioral symptoms typically observed in ASD mouse models to a greater extent than that from neurotypical controls.128 This effect has been ascribed to differential abundance of microbial metabolites, which is commonly observed in ASD,129 leading to altered expression of ASD-linked genes in the brain.128 Conversely, treatment of ASD model mice with normal human commensals such as B. fragilis has been shown to reduce the severity of behavioral symptoms.130 These studies argue in favor of a causative role of microbiota alterations in ASD pathology, both in regulating predisposition via maternal immune activation and in promoting behavioral symptoms in later development, acting as an environmental “hit” in concert with other environmental and genetic factors. Given the abundant microbial metabolite alterations in ASD patients,129 it is feasible that their effects are mediated at least partially via alterations in microglial activity, though to date no studies have confirmed this.MIA pathogenesis is also strongly linked to gut microbiota. Segmented filamentous bacteria (SFB) in the maternal gut are required for induction of the IL-17a response following poly(I:C) exposure, and mice lacking SFB do not show the expected behavioral deficits in MIA offspring.131 Moreover, most studies to date investigating treatment of ASD patients with pre- or probiotic formulations have been described as either inconclusive or unreliable by a recent meta-analysis.110 Despite this, the first trial of fecal microbiota transfer in ASD has reported promising outcomes.133 Thus, the role of both the microbiota and microglia as targets for ASD are still somewhat controversial, but remain an active and promising area of research.In contrast to this idea, one recent study has questioned the causality between gut microbiota composition and ASD, suggesting instead that the behavioral symptoms of ASD drive microbiota alterations via less varied food intake.To date, research into microglial modulation by microbiota has uncovered several promising phenomena of potential relevance to neurological disorders. However, the field is still in its infancy and significant challenges in the interpretation and translation of current findings into disease settings remain to be addressed. The first major caveat to microbiome research in general is that studies in humans largely produce correlative data with little mechanistic insight, and establishing causality between human diseases and microbiome alterations is extremely difficult. This is particularly true of diseases such as AD which exhibit long periods of clinical latency before symptoms manifest. It is for this reason that mouse models are generally relied upon for mechanistic studies seeking to dissect the contribution of microbiota perturbations to disease. However, translating mouse findings back into humans has a notoriously low success rate, which has been attributed not only to issues in preclinical study design, but also to more fundamental flaws in the accuracy with which human diseases are modeled in mice.Germ-free mice, for instance, have provided invaluable insights into the essential role of gut microbes in mammalian biology, but are clearly highly unphysiological and unsuited to modeling the effects of subtle changes to a complex microbiota such as those observed in humans. Many additional gnotobiotic mouse models exist, harboring varying numbers of known bacterial strains, though other than the ASF model their effects on microglial function remain uncharacterized. These models could provide a useful reductionist approach to identify particular bacterial strains which might promote microglial function, either via acetate release or other mechanisms. However, these suffer the same drawback of questionable translational relevance.135 It is also increasingly appreciated that even SPF mice have unnaturally sparse microbiota lacking many natural symbionts and pathogens, which results in relatively immature immune system highly dissimilar to that of humans.136–139 Interestingly, colonization of laboratory mouse strains with complex “wild” biota or infection with pathogens results in a much closer approximation of human immunity than can be achieved in SPF animals.136–139 However, it is currently unclear whether microglial function is affected by colonization with wild biota, or whether its effects relate purely to microbial diversity or rather to the presence of specific immunostimulatory microbes absent from SPF mice. Nonetheless, conventional SPF mice clearly suffer from a paucity of microbial stimulation at baseline, which could render investigation of microbiota-immune interactions prone to artifacts.Theoretically, this limitation can be overcome by use of fecal microbiota derived from human subjects. However, these models should also be interpreted with caution due to various issues including incomplete colonization of the recipient animal and the absence of additional relevant host and environmental factors.Thus, to maximize the impact of microbiota-based therapies on human medicine it is important to establish robust causal relationships in human-relevant models. It would be intriguing for future efforts to establish whether the presence of wild-like microbiota affects microglial function, since this could provide a more robust platform for investigating human-relevant pathways of microbiota-microglia communication and increase the translational success of any resulting strategies. It is equally critical for future studies in human patients to gather high-quality data combining microbiota profiling with metabolomic analyses, ideally including longitudinal study designs and diverse patient cohorts to maximize mechanistic interpretability and identify high-confidence targets for animal model validation. It should also be appreciated that, although direct signaling between microbiota and microglia is a compelling concept, optimal clinical success will likely only come by targeting multiple systems including gut and metabolic function, as well as peripheral immunity.64 Given the great diversity of microbially derived molecules permeating the body, it seems likely that at least some are capable of modulating microglia in ways which remain to be discovered, especially considering that the bulk of studies to date have been performed in a single strain of laboratory mouse under conditions of artificially low microbial diversity. It would therefore be interesting for future studies to dissect the effect of both wild mouse microbiota and transplanted human microbiota on microglial phenotype, with particular focus on the underlying metabolites and mechanisms. Extra care should also be given to examining the relationship between microbiota and microglia at different developmental stages. Embryonic, early postnatal and adult microglia are not only intrinsically different, but also very likely experience divergent sets of microbial cues. Thus, the existence of developmentally specific signaling axes should be considered. Another currently overlooked facet to microbiota research is the role of non-bacterial species such as fungi. Fungi have already been demonstrated to promote social behavior via IL-17 signaling in neurons,140 and to modify Alzheimer’s pathology in APP/PS1 mice via an unclarified pathway.141 Thus, fungi may provide additional routes of microglial modulation by microbiota and are deserving of further study in both humans and mice.Currently, the only robustly defined routes of direct communication between microbiota and microglia occur via acetate and AhR ligands, the latter of which has only been demonstrated in the context of EAE.


Circulating tumor DNA (ctDNA) is a component of cell-free DNA (cfDNA) that is shed by malignant tumors into the bloodstream and other bodily fluids. ctDNA can comprise up to 10% of a patient’s cfDNA depending on their tumor type and burden. The short half-life of ctDNA ensures that its detection captures tumor burden in real-time and offers a non-invasive method of repeatedly evaluating the genomic profile of a patient’s tumor. A challenge in ctDNA detection includes clonal hematopoiesis of indeterminate potential (CHIP), which can be distinguished from tumor variants using a paired whole-blood control. Most assays for ctDNA quantification rely on measurements of somatic variant allele frequency (VAF), which is a mutation-dependent method. Patients with certain types of solid tumors, including colorectal cancer (CRC), can have levels of cfDNA 50 times higher than healthy patients. ctDNA undergoes a precipitous drop shortly after tumor resection and therapy, and rising levels can foreshadow radiologic recurrence on the order of months. The amount of tumor bulk required for ctDNA detection is lower than that for computed tomography (CT) scan detection, with ctDNA detection preceding radiologic recurrence in many cases. cfDNA/ctDNA can be used for tumor molecular profiling to identify resistance mutations when tumor biopsy is not available, to detect minimal residual disease (MRD), to monitor therapy response, and for the detection of tumor relapse. Although ctDNA is not yet implemented in clinical practice, studies are ongoing to define the appropriate way to use it as a tool in the clinic. In this review article, we examine the general aspects of ctDNA, its status as a biomarker, and its role in the management of early (II–III) and late  stage colorectal cancer (CRC). Cell-free DNA (cfDNA) is comprised of double-stranded DNA ranging between 150 and 200 base pairs in length and circulates mostly in blood . Active Circulating tumor DNA (ctDNA) is a component of cfDNA that is shed by malignant tumors into the bloodstream and other bodily fluids, including blood, urine, pleural fluid, ascites, and saliva . In the The amount of cfDNA in cancer patients varies widely, and is usually higher than in the blood of healthy controls . In healIn one study of colorectal cancer (CRC) patients, the mean value of cfDNA was 495.7 ng/mL of plasma (range 100–1750 ng/mL), with a median value of 450 ng/mL . Four moTwo types of information can be obtained through ctDNA analysis: genomic sequences in tumors and the quantification of tumor burden. Quantifying ctDNA at a single time point may allow disease staging and prognosis, and sequence analysis might allow the selection of targeted therapies. ctDNA carries genetic information from the primary and metastatic lesions and can therefore provide insights into clonal heterogeneity and the evolution of the tumor . In contMost of the early data about ctDNA applications in oncology were in the metastatic solid tumor setting for molecular profiling at diagnosis, targeted therapy selection, treatment response monitoring, post-treatment tumor surveillance to detect recurrence, and monitoring treatment resistance  Figure . HoweverIn the neoadjuvant and adjuvant settings, measuring serial ctDNA prior to and throughout treatment may be useful to monitor response to treatment as an early endpoint to potentially predict long-term outcomes . In earlData demonstrated that the amount of tumor bulk required for ctDNA detection is lower than for CT scan detection, with ctDNA detection preceding radiologic recurrence in many cases . This seIn the early stage disease setting, adjuvant trial patient populations are heterogeneous, and several clinical trials are evaluating the potential value of ctDNA to select patients that could benefit from further treatments. The MERMAID-1 (NCT04385368) and MERMAID-2 (NCT04642469) clinical trials are assessing adjuvant treatment with a checkpoint inhibitor in patients with resected stage II and III NSCLC with MRD positive by ctDNA measurements. IMvigor 011 (NCT04660344) for patients with muscle-invasive bladder cancer who are ctDNA positive after cystectomy is exploring the use of ctDNA as a biomarker for patient selection for adjuvant treatment with anti-PDL1 antibodies. These studies are evaluating the use of ctDNA to identify patients at the highest risk of recurrence for enrollment in clinical trials, reducing patient numbers as well as the time and cost of studies.The detection of ctDNA following surgery or treatment with curative intent may signal the presence of MRD, which could identify patients who may be at a higher risk of relapse . Analysip < 0.001, hazard ratio [HR] 14.3, 95% confidence interval [CI] 3.2–64.1) . I. In = 23< 0.001) A. These p < 0.001) (p < 0.001) . Major reductions (>10-fold) versus lesser reduction in ctDNA pre-cycle two were associated with a trend for increased PFS . The optimal criterion for predicting response to therapy was ≥10-fold change in ctDNA after cycle one of chemotherapy. Patients who met this criterion experienced a trend of longer PFS than patients with <10-fold drop in ctDNA . In metastatic CRC, data suggest that ctDNA changes can occur rapidly in response to systemic therapy, with ctDNA variations at 2 weeks being predictive of subsequent radiographic results in restaging studies at 2 months [Tie et al. evaluated 53 mCRC (treatment naïve) patients receiving standard first-line chemotherapy by monitoring ctDNA levels . Tumors 2 months . In conc2 months . No sign2 months . ctDNA m2 months . Another2 months .In conclusion, the results of several studies in CRC consistently demonstrate the potential use of ctDNA as a prognostic tool. The findings of these studies showing the role of ctDNA as a biomarker to detect MRD, follow response to treatment, and surveille disease are summarized in EGFR mutations in the extracellular domain (ECD: amino acids 334 to 505) mediate resistance by blocking the binding of anti-EFGR antibodies . The potential for EGFR ECD mutations to drive resistance to anti-EGFR antibodies is documented through ctDNA and tumor biopsies. However, studies using ctDNA identified a novel cluster of mutations (cluster 1) in EGFR ECD. This previously unreported cluster of EGFR ECD mutations involving V441 and S442 accounted for 25% of all EGFR EDC mutations [Tumor genotype plays an important part in determining drug resistance in patients with mCRC. Genotyping of tumor tissue can help with the selection of patients with tumor amenable to treatment; however, the value of testing a tumor sample is limited by inter- and intra-tumor heterogeneity. Moreover, archival tissue will not show the genotypic changes that occurred since the sample was obtained. However, since tissue-based sequencing compendia rely on early stage and treatment-naïve tumors, these databases have limited insights into acquired resistance mutations. Large ctDNA cohorts can more readily provide non-invasive access to patients with advanced tumors and may offer unique insight into resistance mechanisms emerging under the selective pressure of systemic therapies. The standard treatment for mCRC includes drugs that target the molecular drivers of colorectal cancer pathogenesis, such as VEGF and EGFR ,136. EGFutations .KRAS wild-type tumors, some of whom acquire KRAS mutations during treatment as a mechanism of drug resistance [KRAS alterations as mechanisms of emerging resistance to anti-EGFR therapy in CRC with ctDNA analysis [KRAS resistant subclones 10 months before radiographic progression. Other studies on ctDNA showed that mutated KRAS alleles detected at progression start declining when the anti-EGFR blockage is withdrawn, suggesting a potential role for anti-EGFR re-challenge [The use of anti-EGFR antibody treatment is restricted to patients with sistance ,138. In analysis ,138. Thehallenge ,140. TheClinical trials are already evaluating the potential value of ctDNA in the early stage CRC setting to select patients that could benefit from further treatments. The Australian trial DYNAMIC-II (ACTRN12615000381583) will determine the effect of the use of ctDNA to guide adjuvant chemotherapy in stage II CRC patients. DYNAMIC-III (ACTRN12617001566325) is a phase II/III study enrolling stage III colon cancer patients who will be randomized to be treated according to post-operative ctDNA results (Arm B: ctDNA-informed) or as per the standard of care (Arm A: SOC). Patients who are ctDNA-negative will be managed with a de-escalation adjuvant treatment, and those who are ctDNA-positive will be managed with an escalation adjuvant treatment strategy. In the standard of care arm, patients will receive adjuvant chemotherapy as per the standard of care.The COBRA study (NCT-04068103) is a phase II/III study of ctDNA as a predictive biomarker in adjuvant chemotherapy in patients with stage IIA colon cancer; this study will identify patients with colon cancer after surgery who will benefit and those who will not benefit, from receiving chemotherapy. The IMPROVE-IT trial (NCT03748680) is opened for patients with surgically removed adenocarcinoma of the colon or rectum with pathologically stage I or II diseases, and radical resection with detectable ctDNA in two weeks postoperative plasma sample, and with no indication for adjuvant chemotherapy according to Danish Colorectal Cancer Group (DCCG) guidelines. The primary aim of the study is to investigate if the use of standard adjuvant chemotherapy improves the disease-free survival in patients with MRD detected by ctDNA, where adjuvant chemotherapy is not standard treatment.The Netherlands trial MEDOCC-CrEATE (NL6281/NTR6455) investigates whether adjuvant chemotherapy (ACT) reduces the risk of recurrence in stage II colon cancer patients with detectable ctDNA after surgery .CIRCULATE-Japan is a project that aims to detect MRD and measure treatment responsiveness in resectable CRC using ctDNA testing . CIRCULAThe emerging field of ctDNA research has opened new avenues for cancer diagnostics over the past 5 years, with important clinical opportunities for personalized medicine in oncology. In patients with stages II and III CRC, it was demonstrated that ctDNA may be a useful prognostic marker after surgery and could guide initial adjuvant treatment and could be useful to monitor recurrence. ctDNA analysis can potentially change the postoperative management of CRC by enabling risk stratification, chemotherapy monitoring, and early relapse detection. Several studies in CRC suggested that ctDNA can be used in surveillance and detects recurrence 3–11.5 months earlier than imaging. In the CRC metastatic setting, decreases in ctDNA level during systemic therapy (first or second line of therapy) correlate with tumor response. The clinical use of ctDNA as a biomarker in cancer care will depend on the standardization of pre-analytic and analytic procedures . There a


Belantamab mafodotin (belamaf) is a novel antibody–drug conjugate developed for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). Although the drug has demonstrated a good efficacy, corneal adverse events have been reported. In this prospective study, consecutive patients with RRMM who received belamaf infusions were included. The standard ophthalmological visit was implemented with anterior segment (AS)-optical coherence tomography (OCT) and in vivo confocal microscopy (IVCM). Five patients  with MMRR and unremarkable ocular findings at baseline who received belamaf infusion were included. After a median time of 28 days from the first infusion, four of them developed corneal alterations with transient vision reduction to a variable extent. In particular, corneal deposits of microcyst-like epithelial changes (MECs) were detected centrally in one patient and peripherally in three patients. AS-OCT scans showed a bilateral heterogeneous increase in signal intensity, together with hyper-reflective lesions confined within the epithelium in all cases, except for one case in which they also involved the stroma. Corneal maps showed a transient increase in epithelial thickness in the first phase that was followed by a diffuse decrease in the subsequent phase. IVCM scans showed MECs as hyper-reflective opacities located at the level of corneal epithelium, largely intracellular. Multimodal corneal imaging may implement the current clinical scale, helping us to detect corneal abnormalities in patients under belamaf therapy. This workup provides useful data for monitoring over time corneal findings and for optimizing systemic therapy. Ocular surface diseases are common among hematological patients, either due to the underlying diseases or the therapies employed for their treatment ,2,3. MulBelantamab mafodotin  is a first-in-class antibody–drug conjugate developed for the treatment of patients with RRMM. It consists of an anti-BMCA mAb conjugated to the microtubule inhibitor monomethyl auristatin F. Belamaf eliminates MM cells by multiple mechanisms of action, including apoptosis, antibody-dependent cell-mediated anti-myeloma responses, accompanied by the release of markers characteristic of immunogenic cell death . AlthougHerein, we present a series of patients who developed corneal adverse events associated with belamaf use and describe features, clinical outcomes, and potential importance to oncologists as a harbinger of serious ocular sequelae. Furthermore, a multimodal imaging-based diagnostic workup used in this series to detect and monitor over time corneal alterations is presented.This is a prospective observational study conducted at the Careggi University Hospital  that included consecutive patients who received belamaf infusions at the recommended dose (2.5 mg/kg) every 3 weeks for the treatment of RRMM between January 2021 and March 2021. All patients underwent an ophthalmological visit before starting treatment, including best corrected visual acuity (BCVA) testing, slit lamp evaluation of the cornea with photograph, intraocular pressure measurement and fundoscopy. The standard visit was implemented with a diagnostic workup focused on cornea that included anterior segment (AS)-optical coherence tomography (OCT) for the studying of corneal maps and in vivo confocal microscopy (IVCM). In particular, corneal epithelial thickness was measured in 5 sectors: one central 3 mm diameter and four inner sectors  within a ring (3–6 mm in diameter). Patients were followed-up for at least 12 months and underwent serial ophthalmological visits before each drug infusion.Overall, five patients  with MMRR were screened for belamaf infusion during the study period. At baseline, all of them presented full vision (mean pre-belamaf BCVA of 20/20) and did not present any remarkable corneal findings; thus, they were treated with belamaf according to drug protocol. Following a mean time of 28 days from the first belamaf infusion, four patients developed corneal alterations with a variable degree of vision reduction (mean post-belamaf BCVA of 20/32). On slit lamp examination, corneal deposits with microcyst-like epithelial changes (MECs) were detected bilaterally in the center of the cornea in one patient and in the peripheral cornea in three patients . Of noteAS-OCT scans showed a bilateral heterogeneous increase in signal intensity, together with hyper-reflective lesions corresponding to visible corneal alterations at slit lamp. These lesions were confined within the epithelium, but in one case, they migrated deeper, involving the stroma .Corneal epithelial maps showed a transient CET increase soon after the first infusion, followed by a progressive decrease during the following infusions (“plateau phase”) .Using IVCM, MECs appeared as hyper-reflective opacities, sometimes arranged in clusters, resembling a “pseudo-rosette” pattern, located at the level of corneal epithelium, largely intracellular. In particular, in one patient, multiple hyper-reflective deposits were detected inside corneal epithelium at the level of alar and basal corneal cells rather than superficial cells; in another patient, completely asymptomatic, isolated peripheral sub-epithelial opacities were detected that subsequently were also found at the level of anterior stroma by means of IVCM .Since the introduction of belamaf therapy for RRMM, a number of corneal side effects have been attributed to its use. These mostly include superficial punctate keratopathy and/or MECs detectable at slit lamp examination. Of note, ocular adverse events such as blurred vision, dry-eye-like symptoms and keratopathy have been reported in 69% to 74% of patients in the DREAMM-1 and DREAMM-2 trials, requiring dose reduction or even interruption in 27% to 46% of cases ,9,10.Belamaf-related corneal alterations usually originate in the peripheral region of the cornea and then gradually migrate towards the center  . The locCorneal toxicity is more likely to occur when the drug is used at higher doses, and the proposed mechanism is thought to derive from the off-target effect of its cytotoxic component  .Efforts are being made to understand the etiology behind this clinical entity in order to identify potential mitigation strategies. For this task, multimodal imaging is of crucial importance, as it helps to better characterize the nature of such findings. Of note, the involved areas of corneal epithelium were found to contain hyper-reflective material, rather than microcysts on IVCM. The solid nature of these epithelial lesions is also corroborated by AS-OCT, where they appear once again hyper-reflective. Therefore, this material could potentially represent the accumulation of apoptotic end products in the intercellular spaces of corneal epithelium. Similarly, hyper-reflective lesions on AS-OCT may be the consequence of the accumulation of either pre-apoptotic or degenerated cells caused by the internalization of belantamab . SpecifiIn order to provide a common standardized and repeatable tool for clinical evaluation, an expert board has recently proposed a keratopathy and visual acuity scale  based on the worst finding of either keratopathy subjectively graded on slit lamp examination  or visual acuity testing. Recommendations for the management of corneal events have been formulated according to the KVA scale as follows: continue treatment at the current dose for grade 1/mild events; delay treatment for grade 2/moderate events until the event improves to a grade 1/mild event or resolves, or for grade 3/4 (severe) events, until these improve to a grade 1/mild event .Our report aimed at describing corneal findings of patients receiving belamaf infusions for RRMM, especially focusing on clinical course monitored by different corneal imaging techniques. Thanks to advances in multimodal corneal imaging including IVCM and AS-OCT, ophthalmologists may benefit from these techniques by easily detecting corneal abnormalities, even at a subclinical stage, and subsequently defining and monitoring over time the entity of corneal involvement ,12,13,14Using IVCM, MECs appear as hyper-reflective opacities, sometimes arranged in a “pseudo-rosette” pattern, located within the basal epithelium and the sub-basal nerve plexus; the stroma shows only non-specific signs of inflammation with sporadic activated keratocytes, while the endothelium is typically unaffected; specific lesions at the sub-basal nerve plexus layer were also recently detected and characterized . HoweverHowever, it should be pointed out that, since half of the patients may present objective signs of keratopathy without reporting any symptoms, ophthalmic examination should be performed at baseline and before each dose of the drug in order to detect early corneal changes.The management of belamaf-induced toxicity requires a multidisciplinary approach involving a hematologist and an ophthalmologist, and the clinical decision should be reached together taking into account the individual benefit/risk ratio according to both ocular and systemic conditions. Overall, single-agent belamaf 2.5 mg/kg) has shown a manageable safety profile. Ocular alterations usually recover spontaneously once treatment is discontinued or may be successfully reversed with dose tapering or by delaying the time interval of suspension between two cycles. This evidence is based on the experience of a DREAMM-2 study that reported the need for drug discontinuation due to keratopathy in only 1 out 95 patients who received the 2.5 mg/kg dose, indicating that patients were able to remain on treatment while these events were monitored  mg/kg ha. The impFuture prospective trials are needed to provide more comprehensive information about belamaf-associated keratopathy as well as for identifying the most appropriate diagnostic workup able to optimize therapy in RRMM patients.


Nematostella vectensis (Anthozoa), but little is known about the evolutionary origins of this channel diversity and its conservation across Cnidaria. Here, we examined the evolution of voltage-gated K+ channels in Cnidaria by comparing genomes and transcriptomes of diverse cnidarian species from Anthozoa and Medusozoa. We found an average of over 40 voltage-gated K+ channel genes per species, and a phylogenetic reconstruction of the Kv, KCNQ, and Ether-a-go-go (EAG) gene families identified 28 voltage-gated K+ channels present in the last common ancestor of Anthozoa and Medusozoa . Thus, much of the diversification of these channels took place in the stem cnidarian lineage prior to the emergence of modern cnidarian classes. In contrast, the stem bilaterian lineage, from which humans evolved, contained no more than nine voltage-gated K+ channels. These results hint at a complexity to electrical signaling in all cnidarians that contrasts with the perceived anatomical simplicity of their neuromuscular systems. These data provide a foundation from which the function of these cnidarian channels can be investigated, which will undoubtedly provide important insights into cnidarian physiology.Ion channels are highly diverse in the cnidarian model organism  Nematostella vectensis revealed a surprising diversity of ion channels, raising the possibility that cnidarians might have complex electrical signaling despite having outwardly simple neuromuscular anatomy. Here, we show that all cnidarians have a high diversity of voltage-gated K+ channels, and we find evidence for 28 of these channels in stem cnidarians. This contrasts sharply with nine voltage-gated K+ channels predicted for stem bilaterians and suggests that unexpectedly diversified electrical signaling properties are a central requirement for cnidarian physiology.The genome of the starlet sea anemone + channels comprise three highly conserved and molecularly distinct gene families, Shaker (Kv), KCNQ, and Ether-a-go-go (EAG). These channels fill a wide range of physiological roles, most notably regulating neuronal and muscular excitability by controlling action potential threshold and repolarization. All three voltage-gated K+ gene families encode structurally homologous tetrameric channels, with each subunit contributing one-fourth of a tetrameric pore domain (PD) and one of four independent voltage sensor domains (VSDs) that surround the PD  attached to the pore via a helical domain known as the C-linker  which is evolutionarily related to BTB/POZ dimerization domains found in some transcription factors (C) which appears to play a similar role as T1 in channel assembly  to bilaterian channels indicates that eight of the nine predicted bilaterian ancestral channels were also present in the cnidarian/bilaterian ancestor. Nematostella contains a single KCNQ paralog that is an outgroup to the two types of bilaterian KCNQs  whose last common ancestor is believed to be the ancestor of all extant cnidarians  to provide a relatively high resolution view of evolutionary events that led to the extensive channel set of the highly developed experimental model N. vectensis  to gain insights into the channel set of the last common anthozoan ancestor. The inclusion of two schyphozoans  and a hydrozoan  provided us with insight into channel ancestry and diversification within the medusozoan lineage. We did not include Endocnidozoa in our study because they derive from the same common ancestor as Anthozoa and Hydrozoa  as had been effectively applied to identify K+ and other channels in previous studies  and Drosophila (9) and similar to vertebrates such as humans (40)  . Note weme draft  comparedme draft . All cniN. vectensis, A. digitifera, and Orbicella faveolata) but six KCNQs in H. vulgaris. Here, we identified a seventh KCNQ channel in H. vulgaris that was previously missed. Our phylogeny of the KCNQ family is consistent with there being a single KCNQ gene in the last common ancestor of Cnidaria . In contrast, our phylogeny is consistent with a KCNQ duplication in the medusozoan lineage prior to the divergence of scyphozoans and hydrozoans to produce two ortholog groups , whereas KCNQ2 duplicated twice in the lineage leading to Scyphozoa (KCNQ2a–c). Four additional Hydra KCNQ channels (KCNQ3a–d) form a separate clade in the phylogeny. Unlike all other KCNQ channels, these KCNQ3s share an intron-less gene structure across the conserved core suggesting they are the product of a relatively recent retrotransposition event.Cnidaria . In addiRe. muelleri lineage and three times in the H. vulgaris lineage  or absence (Nverg2-5) of the eag gating domain , 5 within Hexacorallia , 4 in anemones (present in Nematostella and Exaiptasia), and only 1 in the Nematostella lineage after it diverged from Exaiptasia  to look for orthologs of missing ancestral channels in Hydrozoa and Octocorallia, and (2) to get a preliminary read on voltage-gated K+ channel diversity in two additional medusozoan classes, Staurozoa and Cubozoa. Note this broader supplementary search was designed simply to look for the presence of ancestral voltage-gated K+ channel in various cnidarian classes and was not intended to provide insight into the full channel complement of additional species. Kv, KCNQ, and Eag phylogenies containing voltage-gated K+ channel sequences from additional cnidarian species (listed in + channels in Hydrozoa (including a H. vulgaris ShawR4 ortholog that we had previously missed), but no evidence for additional ancestral sequences in Octocorallia  in D. melanogaster  and medusozoan (hydrozoan) voltage-gated K+ channel orthologs. NvShak1  cnidarian and bilaterian channels branch separately in phylogenies  Kv, EAG, and KCNQ sequences as queries  plus the T1 domain for Kvs, the coiled-coil C-terminal tail for KCNQ, or the EAG/PAS and CNBD domains in EAG channels (excluding Erg subfamily genes lacking the EAG domain and certain H. vulgaris Eag sequences). We used “complete” Kv sequences as queries for reciprocal best BLASTP searches against the N. vectensis protein database and used “complete” EAG and KCNQ sequences as queries for reciprocal best BLASTP searches against Mus musculus Refseq , A. digcorallia ), E. palcorallia ), Re. mucorallia ), H. vulHydrozoa ), Sa. macyphozoa ), Rh. escyphozoa ). We dess Refseq . Hits wiWe generated alignments for our main phylogenetic analyses using MUSCLE  as imple+ channel genes, we identified the smallest clades containing genes from at least one anthozoan and medusozoan species in each of the three phylogenies. We only considered clades with posterior probabilities>0.95, excepting the NvShak6 clade, which had a posterior probability of 0.93. All sequences used in the phylogenies are provided in We constructed individual phylogenies for each gene family via Bayesian inference using MrBayes (v3.2.7a)  with BEAevad009_Supplementary_DataClick here for additional data file.


Instead, we developed a replacement assay that used non-lethal dose-response methods to assess the sensitivity of 12 wildlife species and laboratory rats to PAPP that did not require death to be used as an experimental outcome. By establishing the relationship between non-lethal doses of PAPP and the formation of methaemoglobin (MetHb), we found that we could accurately predict doses likely to be lethal. Our estimates very closely approximated existing LD50 values determined for PAPP. We argue that laboratory-based lethal-dose bioassays are unsuited to assessing the comparative hazard of toxicants to wildlife species. In contrast, non-lethal assays that use biologically relevant measures can provide much more robust and meaningful indications of relative hazard even in species of high conservation value, where lethal experimentation can rarely be justified.Para-aminopropiophenone (PAPP) is registered as bait poison for the humane control of red foxes and wild dogs in Australia. To classify the relative hazard of poisons, regulatory bodies have historically demanded LD80). The comparative sensitivity of non-target mammals referenced to this endpoint was found to be highly variable, with southern brown bandicoots (Isoodon obesulus) the most sensitive species (MetHb80 = 6.3 mg kg−1) and bush rats (Rattus fuscipes) the most tolerant (MetHb80 = 1035 mg kg−1). Published LD50 estimates were highly correlated with PAPP doses modelled to achieve the MetHb80 endpoint . Most dose-response data for native mammals were collected in the field or in semi-natural enclosures, permitting PAPP and placebo dosed animals to be fitted with tracking transmitters and transponders and released at their point of capture. A protracted morbidity and mortality was observed only in Australian ravens (Corvus coronoides). The combination of sub-lethal dose-response assay and survival data collected in the field provided more relevant information about the actual hazard of pest control agents to non-target wildlife species than laboratory-based lethal-dose bioassays. We discuss the need to replace lethal-dose data with biologically meaningful insights able to define a continuum of toxicological hazards that better serve the needs of conservation and veterinary scientists and wildlife managers.Para-aminopropiophenone (PAPP) is a potent methaemoglobin (MetHb) forming agent used for the lethal control of exotic carnivores and mustelids. To assess the sensitivity of Australian wildlife to PAPP we developed an in vivo assay that did not use death as an endpoint. Sub-lethal dose-response data were modelled to predict PAPP doses required to achieve an endpoint set at 80% MetHb (MetHb Felis catus) and red foxes (Vulpes vulpes)  ,42. The 80 = 6.3–1035 mg kg−1). Modelling dose-dependent MetHb responses permitted MetHb80 endpoints to be predicted, that were strongly correlated with known LD50 values for five species. Survival data were collected in the field, avoiding the need for captivity in a laboratory setting where animals would be subjected to a range of novel stressors. Survival data collected over a 10-day period allowed protracted effects upon Australian ravens to be identified. Overall, sub-lethal dose-response methods were a viable alternative to lethal-dose bioassays for establishing the comparative hazard of PAPP to Australian wildlife.Australian mammals were found to vary greatly in their comparative sensitivity to PAPP (MetHbLethal dose bioassays measure only dichotomous data—death and survival. In contrast, assays that monitor continuous variables linked to dose-dependent pathophysiological changes may be correlated with changing animal welfare states. Non-lethal assays potentially allow thresholds to be set to demarcate acceptable and unacceptable welfare impacts associated with perturbations in pathophysiological markers at levels well below those that cause mortality. Non-lethal assays ensure that sensitivity data can be collected in a wide range of species, including high conservation value species, where lethal experimentation can rarely be justified.


Introduction: Diamond Blackfan anemia (DBA) is an autosomal dominant ribosomopathy caused predominantly by pathogenic germline variants in ribosomal protein genes. It is characterized by failure of red blood cell production, and common features include congenital malformations and cancer predisposition. Mainstays of treatment are corticosteroids, red blood cell transfusions, and hematologic stem cell transplantation (HSCT). Despite a better understanding of the genotype of DBA, the biological mechanism resulting in the clinical phenotype remains poorly understood, and wide heterogeneity can be seen even within a single family as depicted here.Case Description: Thirty family members enrolled in the National Cancer Institute inherited bone marrow failure syndromes study were evaluated with detailed medical questionnaires and physical examinations, including 22 in the family bloodline and eight unrelated partners. Eight participants had been previously told they had DBA by clinical criteria. Targeted germline RPS19 testing was done on all family members. A pathogenic heterozygous missense mutation in RPS19  was detected in ten family members, including one person previously presumed unaffected. Eight family members presented with macrocytic anemia in infancy; all of whom were responsive to prednisone. Four family members became treatment independent; however, one individual became transfusion-dependent 36 years later following an episode of pneumonia. One prednisone responsive individual electively discontinued steroid treatment, and lives with severe anemia. One prednisone responsive individual died at age 28 from a stroke. Two family members developed colorectal cancer in their fifties; one had never required treatment for anemia. None had major congenital anomalies.Discussion: This large family with DBA demonstrates the heterogeneity of phenotypes that can be seen within the same genotype. Most family members presented with steroid-responsive anemia in infancy and subtle congenital malformations, findings consistent with recent genotype-phenotype studies of RPS DBA. However, two family members were relatively unaffected, underscoring the importance of further studies to assess modifier genes, and epigenetic and/or environmental factors which may result in normal erythropoiesis despite underlying ribosome dysfunction. This large, multigenerational family highlights the need for individualized treatment, the importance of early cancer surveillance even in individuals with clinically mild phenotypes, and the benefit of long-term follow-up to identify late complications. TSR2 and GATA1, which encode a ribosome chaperone and a hematopoietic transcription factor targeted by altered ribosome levels (RPS19) is the most commonly affected gene and accounts for approximately 25% of all DBA cases (Diamond Blackfan anemia (DBA) is predominantly an autosomal dominant inherited red cell aplasia caused by deleterious mutations in ribosomal genes. To date, over 200 mutations in one of 26 ribosomal proteins (RP) affecting the large (RPL) or small (RPS) subunit have been identified as well as two X-linked genes, e levels . RibosomBA cases .As a prototypical ribosomopathy, DBA demonstrates an incongruous transition from early symptoms due to cellular hypo-proliferation to cellular hyper-proliferation with an elevated cancer risk later in life . The damDBA is characterized by failure of red blood cell production, congenital abnormalities, poor linear growth, and cancer predisposition . HoweverRPS19. The clinical presentation of some family members was previously reported over 30 years years ago (www.marrowfailure.cancer.gov).Despite a better understanding of the underlying germline genetic and ribosomal etiology of DBA, the biological mechanism resulting in variable penetrance, wide spectrum of phenotypes, and whether individuals with clinically mild phenotypes carry a life-long cancer predisposition, is still unclear. Genotype-phenotype correlations have been studied but are difficult due to the rarity of DBA, heterogenous genotype, and need for long term follow-up for many of the outcomes of interest such as cancer . Here, wears ago . We now n = 10; n = 20) are participants in the IBMFS study at the NCI, an ongoing natural history and retrospective/prospective longitudinal cohort study approved by the NCI Institutional Review Board   was previously identified. The family initially enrolled in the study in 2003 and the most recent follow-up data are from 2022. The age at last follow-up was obtained for all subjects with the date of death serving as the age at last follow-up for those who were deceased.All DBA cases n = 10;  and thei0027274) . Each paRPS19  was detected in ten family members, including one individual who was entirely asymptomatic and previously presumed unaffected  became transfusion-dependent 36 years later following an episode of pneumonia. His treatment complications include severe glaucoma and iron overload. He is enrolled in the NIH clinical trial “Treatment of Refractory DBA with Eltrombopag” (NCT04269889) , a trialNone of the individuals in this family with DBA had significant cardiac, craniofacial, genitourinary or limb anomalies. Seven individuals with DBA had hypoplastic thenar muscles. Six individuals had short stature, defined by < 10th percentile for height based on age and sex, in comparison to only one individual in this family without DBA. One individual was born with spina bifid occulta (50-4) and another family member had mild dysmorphic features, developmental delays and congenital heart block (50-3).Two family members developed colorectal cancer (CRC) at 56 and 61 years of age, one of whom (50-6) had never required treatment for anemia and was only mildly anemic during pregnancy. She underwent surgical resection of her CRC, has completed radiation therapy, and is nearing completion of chemotherapy which she tolerated well without increased blood cell count suppression. The proband (50-1) had been treatment-independent for over 50 years. He underwent a colonoscopy upon CRC diagnosis in his sibling (50-6) and was identified to have localized CRC at 61 years of age which was resected. He tolerated his chemotherapy well and only required one pRBC transfusion when his hemoglobin dropped to 7.9 g/dL after his eighth and last cycle of chemotherapy. Currently he is 5 months off treatment for his CRC and he remains treatment independent from a DBA standpoint.As a classic ribosomopathy, DBA illustrates how intricate cellular complexities involved in ribosome biogenesis can result in a spectrum of phenotypic abnormalities in patients with DBA. However, the factors that modulate individual responses to ribosome dysfunction remain unclear. This large, multigenerational family demonstrates the spectrum of variable penetrance and expressivity that can be seen within the same DBA genotype as well as the importance of new targeted therapeutics in this heterogeneous disease. This family additionally provides insight into cancer predisposition even for those with mild phenotypes and the importance of long-term follow-up to identify late complications in DBA.RPS19 and RPS vs. RPL-DBA , and ample time for patients to develop disease and/or treatment-related complications. However, this approach does have some limitations in that we require clinical updates through family questionnaires and clinical records from primary providers, which can lead to some incomplete information. For instance, most clinical details surrounding the death of patient 50-3 are unknown and thus it is unclear if her stroke may have been related to long term corticosteroid use. Additionally, patient 50-8 has not submitted clinical records since age 11 years, so it is unclear whether she has remained truly asymptomatic. Despite these inevitable challenges of natural history studies, they are still of utmost importance in advancing the understanding of rare diseases. In conclusion, DBA may be a different disease in every patient owing to the effects of genetic background and environment. DBA natural history studies are important to help elucidate yet unidentified factors that regulate phenotypic expression of a ribosomal genetic defect. Longitudinal follow-up is important to provide an understanding of factors that may potentiate treatment dependence or independence, and insight into cancer predisposition for those with clinically mild phenotypes. An understanding of factors influencing clinically milder phenotypes may provide insights into the pathophysiology of this ribosomopathy and ultimately lead to improved treatment options.Families tend to enroll in the IBMFS at the NCI to better understand the natural history of their disease and to have access to additional scientific and clinical expertise in DBA. Over the years, we have met the family in person for clinical evaluation and genetic testing and have also had countless emails and phone calls to discuss anything from DBA summer camp options to second opinions on cancer treatment.


Patients with end-stage kidney disease (ESKD) wait roughly 4 years for a kidney transplant. A potential way to reduce wait times is using hepatitis C virus (HCV)–viremic kidneys.As preparation for developing a shared decision-making tool to assist patients with ESKD with the decision to accept an HCV-viremic kidney transplant, our initial goal was to assess the feasibility of using The Gambler II, a health utility assessment tool, in an ambulatory dialysis clinic setting. Our secondary goals were to collect health utilities for patients with ESKD and to explore whether the use of race-matched versus race-mismatched exemplars impacted the knowledge gained during the assessment process.We used The Gambler II to elicit utilities for the following ESKD-related health states: hemodialysis, kidney transplant with HCV-unexposed kidney, and transplantation with HCV-viremic kidney. We created race exemplar video clips describing these health states and randomly assigned patients into the race-matched or race-mismatched video arms. We obtained utilities for these 3 health states from each patient, and we evaluated knowledge about ESKD and HCV-associated health conditions with pre- and postintervention knowledge assessments.P<.05) following utility assessment process. The use of race-matched exemplars had little effect on the results of the knowledge assessment of patients.A total of 63 patients with hemodialysis from 4 outpatient Dialysis Center Inc sites completed the study. Mean adjusted standard gamble utilities for hemodialysis, transplant with HCV-unexposed kidney, and transplantation with HCV-viremic kidney were 82.5, 89, and 75.5, respectively. General group knowledge assessment scores improved by 10 points (Using The Gambler II to collect utilities for patients with ESKD in an ambulatory dialysis clinic setting proved feasible. In addition, educational information about health states provided as part of the utility assessment process tool improved patients’ knowledge and understanding about ESKD-related health states and implications of organ transplantation with HCV-viremic kidneys. A wide variation in patient health state utilities reinforces the importance of incorporating patients’ preferences into decisions regarding use of HCV-viremic kidneys for transplantation. Chronic kidney disease disproportionally impacts African Americans, making it a prototypical disease in which to investigate disparities in health utility assessments (HUAs) . Due to As of 2017, a total of 746,557 patients in the United States have ESKD . A totalThe Gambler II is a health utility assessment software platform that can use a variety of assessment techniques to gather patients’ utilities ,11. ThesOur longer-term goal is to develop an SDM tool that can be used by clinicians to facilitate discussions about acceptance of HCV-viremic kidneys, particularly for patients who may have longer predicted waiting times on transplant lists. The tool would assess patients’ utilities for hemodialysis and transplantation with either an HCV-unexposed or an HCV-viremic kidney and would use that information along with patient-specific demographic information and predictions of organ availability based on factors including age, sex, blood type, dialysis vintage, calculated panel reactive antibodies, comorbidities, and the region in which the transplant is being carried out to make a recommendation for the best transplantation strategy for that patient. As many patients on dialysis, particularly African Americans living in less affluent urban areas, are impacted by the digital divide and have less access to home computers and the internet , we enviThis study was reviewed and approved by the University of Cincinnati Institutional Review Board (UC IRB ID – 2019-0792) as well as by the Dialysis Clinic Inc Administrative Review Office. Both boards approved the study before patient recruitment began. No compensation was provided to the patients for this research.We worked with physicians in the Division of Nephrology and Hypertension to help recruit patients with chronic hemodialysis receiving treatment at any of the 4 outpatient dialysis centers in the greater Cincinnati metropolitan area managed by Dialysis Center Inc (DCI) . InclusiPatients were recruited from 4 DCI sites in the Cincinnati metropolitan region. Prior to participating in the utility assessment process, they underwent a knowledge assessment survey. We collected demographic information and assessed health literacy using the Rapid Estimate of Adult Literacy in Medicine and subjective numeracy. We randomized patients to 1 of 2 study arms in which they viewed either race-matched or race-mismatched video clips describing each of the 3 health states. The patients’ utilities for these health states were obtained, and then a postinterview knowledge assessment was repeated . Those pt test. We randomized patients into 1 of 2 study arms , highest educational level attained, dialysis vintage, history of prior kidney transplant, interest in receiving a transplanted kidney, health literacy using the Rapid Estimate of Adult Literacy in Medicine Short Form, and subjective numeracy -22.We conducted surveys and performed HUAs using a laptop computer (2014 Apple MacBook Pro running Mac OS 10.14) while we met with most patients in their community dialysis center during their usual intermittent hemodialysis sessions. Patients used either headphones, earbuds, or audio output from the laptop to hear the video clips that were part of The Gambler II. We used Google Chrome version 80 as the web browser to access The Gambler II and REDCap (9.1.10). Patient actors used a standard script for each health state, which was developed and vetted by team members . We recorded 12 different video clips describing the 3 health states for all combinations of gender and race  using Apple’s QuickTime X 10.5 with video mastering done on Adobe Premiere and Adobe Audition 2019. Due to restrictions of social distancing during the COVID-19 pandemic, the institutional review board granted an amendment to the protocol that allowed us to interview some patients remotely . For theWe used The Gambler II to perform health utility assessments . For surP=.07; We recruited 71 patients from 4 DCI sites in the Cincinnati Metropolitan area. A total of 63 patients consented and completed the study. Of those who consented, 62 (98%) enrolled in the study during dialysis clinic visits, while 1 (2%) enrolled through teleconferencing software at their residence. The cohort’s age range was from 24 to 80 years, with a median age of 59 years and mean age of 58 years . MoreoveP<.001, paired Wilcoxon t test). However, there was not a statistically significant difference in pretest versus posttest change scores between the 2 study arms, or between African Americans compared with European Americans, with P values of .95 and .96 (Mann-Whitney test), respectively.We conducted a knowledge assessment using a 10-item questionnaire administered before the utility assessment as a pretest and after as a posttest . The CroWe assessed utilities from patients on chronic intermittent hemodialysis for 3 health states relevant to decision-making about kidney transplantation. The results are reported in P=.04 [Mann-Whitney]).Utility assessments for transplantation with an HCV-infected kidney were carried out with anchor states of transplant with HCV-unexposed kidney as the best outcome and death as the worst outcome. We normalized these utilities to the same 0-100 scale used for the other health states by multiplying the raw utility for transplant with HCV-exposed kidney times the utility of transplant with an HCV-unexposed kidney. Normalized utility means using VAS, SG, and TTO assessments were 66.3, 75.5, and 73.8, respectively. Looking at the raw SG utility weights for transplantation with an HCV-viremic kidney, given a choice between transplantation with an HCV-unexposed kidney and an HCV-viremic kidney, patients were willing to take a 24.5% chance of dying to avoid receiving an HCV-viremic kidney. We did not find statistically significant differences in utilities between race-matched and race-mismatched study arms. However, we did find that African Americans on average had higher utility weights than European Americans for hemodialysis evaluated with the TTO .Our second goal was to collect health utilities from patients with dialysis for several ESKD health states relevant to the decision about kidney transplantation. We found a wide variation in health utilities from patient to patient. Consistent with prior studies, utilities assessed using the SG technique were higher than those determined with the VAS or TTO techniques. SG holistically incorporates risk attitude into its assessment, and since most people are risk averse, SG utilities tend to be higher . Of noteP=.004) during the pandemic. We did not see similar differences among patients in the prepandemic cohort given the small sample size of European Americas (n≤5).During the testing of The Gambler II in a clinical setting, the COVID-19 pandemic interrupted patient recruitment. We performed a subanalysis on patients who enrolled after the COVID-19 interruption. We found that gender and study arm did not have a statistically significant impact on knowledge scores or utilities. Stratifying by race, we found that African Americans had higher SG utilities for all but hemodialysis . FurtherP=.29). However, our cohort only had 9 (14%) patients with a prior transplant.To our knowledge, this is the first paper directly eliciting health utilities from patients with dialysis for transplant with an HCV-viremic kidney. Our major finding is that patients’ utilities for this outcome vary dramatically, and that for some patients, the worry and concern associated with even a successful transplant of an HCV-viremic kidney may result in a utility weight lower than their current health state of chronic intermittent hemodialysis. We found a slight negative correlation between the difference between the SG utility for transplant with an HCV-viremic kidney and hemodialysis and dialysis vintage. For patients who have a longer dialysis vintage, SG utility for transplant with an HCV-viremic kidney and hemodialysis had a negative correlation (–0.16). This means that there is a trend toward a more negative view of transplant with an HCV-viremic kidney compared with hemodialysis among patients who have been on dialysis for a longer time. We also examined how patients’ history of a failed kidney transplant may affect health utilities. We compared this patient population hemodialysis and transplant with an HCV-viremic kidney SG utilities. There was a compelling but not statistically significant trend toward a larger decrement in this value among patients with prior failed kidney transplant .Regarding our third goal, our study showed that health state videos narrated by patient actors, viewed as part of the process of assessing patients’ values and preferences for health outcomes of ESKD, can improve knowledge about these health states. On average, knowledge scores improved by 10 points between the pretest and posttest, demonstrating that these videos can educate patients about the relevant health states as a beneficial side effect of the utility assessment process. While we expected to find a positive impact of using race-matched exemplars compared with race-mismatched exemplars, there were no significant differences between study arms on knowledge gain. Whether our study simply lacked the power to detect a difference and whether matching patient demographics truly matters is a question for further investigation. Of note, patients watching videos narrated by African American patient actors had greater knowledge gains (median 10.0) than those viewing videos narrated by European American patient actors (median 5.0), although this was not statistically significant (Regardless, patients found the videos informative and impactful. Following the utility assessment process, several patients voiced their appreciation for the video descriptions of health states. Some patients even reported being moved to tears.Prior studies noted the potential benefits of using HCV-viremic kidneys to expand the pool of organs available to hemodialysis patients, thus reducing waiting times for transplantation and providing opportunities for transplantation in patients who might not have received a kidney otherwise. Cost-effectiveness analyses have shown the strategy to be cost-effective at a policy level for the general population of patients with ESKD ,22. HoweThe Gambler II provides a consistent and efficient platform to elicit patient utilities and could be integrated into a tool to facilitate shared decision-making. One could envision such a tool that performs personalized decision analyses, using a combination of individual patient’s utilities along with clinical and demographic information needed to estimate organ waiting list times, to provide estimates of quality-adjusted survival or life expectancy with each strategy for that individual patient.Our study had some limitations. Given the demographics of the Cincinnati metropolitan area dialysis clinics in our study, it was difficult to recruit patients with European American racial backgrounds in equal numbers to those with African American backgrounds. In addition, the COVID-19 pandemic negatively impacted our total recruitment due to a pause in study activities between March and October of 2020 . A possiIn conclusion, we found that it is feasible to use a computer platform to assess patients’ utilities for health states related to ESKD in ambulatory community dialysis clinics. Furthermore, we demonstrated that utility weights vary dramatically from person to person. These findings have implications for the future development of shared decision-making tools to aid clinicians and their patients with the challenging question of whether to accept transplantation with HCV-viremic kidneys in patients to reduce waiting times and decrease time spent on hemodialysis. This decision will depend upon both expected organ–waiting list times for HCV-unexposed and HCV-exposed kidneys and individual patient’s values and preferences for these relevant health states.