Influenza vaccines have been available for over 80 years. They have contributed to significant reductions in influenza morbidity and mortality. However, there have been limitations in their effectiveness, in part due to the continuous antigenic evolution of seasonal influenza viruses, but also due to the predominant use of embryonated chicken eggs for their production. The latter furthermore limits their worldwide production timelines and scale. Therefore today, alternative approaches for their design and production are increasingly pursued, with already licensed quadrivalent seasonal influenza vaccines produced in cell cultures, including based on a baculovirus expression system. Next-generation influenza vaccines aim at inducing broader and longer-lasting immune responses to overcome seasonal influenza virus antigenic drift and to timely address the emergence of a new pandemic influenza virus. Tailored approaches target mechanisms to improve vaccine-induced immune responses in individuals with a weakened immune system, in particular older adults. Influenza viruses have been recognized as the causative agents of significant respiratory infections in humans for centuries, with recorded pandemics being described as early as the 16th century . An estiInfluenza viruses are members of the Orthomyxoviridae family. These are enveloped RNA viruses with a segmented genome of 8 single-stranded negative-sense RNA segments. These encode the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA), the nucleoprotein (NP), the matrix protein (M1) and ion channel protein (M2), the polymerase subunits , the nonstructural protein (NS1), the nuclear export protein (NEP), and the more recently discovered PB1-F2, PB1 N40, PA-X, and M42 proteins . While iZoonotic infections from domestic pigs and poultry have caused numerous human influenza cases, which usually are not or only poorly transmissible between humans. Human infections with H5 HPAIV, H7 LPAIV, H7 HPAIV, and H9 LPAIV of poultry have caused many cases of influenza with high fatality rates in recent years. The further adaptation of these viruses to replication in, and transmission among, humans may lead to the eventual development of a pandemic virus. Four influenza pandemics have emerged in the past century, causing the Spanish flu , the Asian flu , the Hong Kong flu , and the swine-origin flu pandemic of 2009 (H1N1/09). Although these viruses eventually have all originated from wild avian reservoirs, new genetically reassorted viruses typically emerged in domestic pigs and poultry, before initiating pandemics in humans.Upon their introduction, pandemic viruses can spread rapidly and circulate among a virtually naive and susceptible human population that has not been previously exposed to an antigenically similar influenza A virus, often resulting in devastating morbidity and mortality. Collectively, the 4 pandemics of the last century have resulted in more than 50 million human fatalities. These pandemic viruses all have continued to circulate after their respective pandemics were over and typically replaced one of the previously circulating seasonal influenza A viruses. The new seasonal influenza viruses gradually drift genetically and antigenically, escaping from antibody-mediated virus neutralizing immunity that builds up in the population upon their annual reappearance. The virus neutralizing immunity that drives seasonal influenza genetic and antigenic drift appears predominantly directed against the globular head of the virus HA protein. As a result, this glycoprotein continuously accumulates mutations that eventually affect recognition by the existing neutralizing antibody landscape across the population. The continuous antigenic drift of seasonal influenza viruses requires regular updates of seasonal influenza vaccines in order to correctly match the circulating viruses .In the interpandemic periods, annual seasonal influenza epidemics, which typically occur in and around winter months in temperate climate zones, collectively have resulted in at least an equivalent number of fatal cases as the 4 past pandemics combined. Severe seasonal influenza infections and complications mainly occur in the so-called high-risk groups, such as older adults, people with chronic disease or impaired immunity, pregnant women, and young children. Therefore, these groups are the first targets of annual influenza vaccination programs, as advised by the World Health Organization (WHO) as well as national and regional public health agencies, such as the US Center for Disease Control and Prevention (CDC), although their response to vaccination is often suboptimal. Because of their contacts with high-risk groups, health care workers also are advised to be annually vaccinated against seasonal influenza.An estimated 2%–10% of vaccinated, healthy individuals do not produce adequate levels of antibodies following vaccination . This maNevertheless, vaccination is the most cost-effective way to prevent influenza virus infections. Until recently, all influenza vaccines were generated in embryonated chicken eggs, based on a technology that was developed in the middle of the 20th century. Although this proved to be a quite efficient method to produce high virus concentrations, it has shown major shortcomings calling for new generation influenza vaccines. Currently available seasonal influenza vaccines are egg- and cell-based inactivated influenza vaccines (IIVs), a live attenuated influenza vaccine (LAIV), and a baculovirus recombinant HA vaccine that is produced in insect cells . The devThe history of influenza vaccination is a success story that started almost a century ago . The firRemarkable advances in our understanding of the correlates of immune protection against influenza increasingly point towards the possible development of improved and more broadly protective vaccines against seasonal and pandemic influenza. In addition to animal and cell culture-based models of influenza, human challenge studies have made significant contributions to the identification of correlates of protection . A numbIt is generally accepted that antibodies directed against the globular head domain of the influenza virus HA are a major correlate of protection. The HA is the viral receptor binding protein allowing attachment of virus particles to cellular targets, before endocytosis, fusion of viral and cellular membranes, and actual infection occur. Antibodies directed to epitopes located in or in close proximity to the receptor-binding site (RBD) can prevent binding of the virus to its receptor, thereby neutralizing the virus before cellular infection takes place. When serum titers of virus strain-specific HA antibodies, induced by vaccination or infection, are high enough, they protect subjects from subsequent (re)infection. The protective effect of virus-neutralizing HA-specific antibodies has been demonstrated both in experimentally infected animals and humans . The indFor optimal vaccine efficacy, it is essential that the vaccine strains antigenically match the epidemic strains closely. Because the HA of seasonal influenza virus strains undergo continuous antigenic drift, seasonal influenza viruses eventually evolve to escape from recognition by virus-neutralizing antibodies, necessitating the update of the vaccine strains almost annually , 52.The NA is the other major viral envelope protein and functions as a receptor-destroying enzyme, which is crucial for efficient release of virus from infected cells during the last stages of the virus replication cycle. Antibodies directed against the enzymatic site of NA can block its function and can contribute to protective immunity. This has been demonstrated in various in vitro and in vivo systems . In contOther antibodies that may contribute to broadly protective immunity include those directed against conserved proteins like M2 , NP, NA The M2 protein is a minor antigen on virus particles but is abundantly expressed on virus-infected cells. Compared to HA and NA, M2 is poorly immunogenic because antisera raised against the virus typically contain few M2-specific antibodies. The protective effect of M2-specific antibodies has been demonstrated after hyperimmunization and passive administration of these antibodies in animal models  and was + T cells. Therefore, it has been suggested that formation of NP immune complexes and opsonization play a role in protection [A protective effect of NP-specific antibodies has been demonstrated in mice , 77, altotection , 77, altotection .The identification of virus-neutralizing antibodies directed to the stalk region of the trimeric HA molecule, has attracted a lot of attention , becauseThe production of polymeric immunoglobulin A (pIgA) and the subsequent transcytosis across the epithelium after binding to the polymeric Ig receptor (pIgR) yields secretory IgA (sIgA), a complex consisting of dimeric IgA and the secretory component, which is a cleavage product of the pIgR .sIgA is more efficient than IgG or monomeric IgA for inhibiting influenza virus entry . Most va+ T cells, are an important correlate of protection against influenza virus infections and contribute to heterosubtypic immunity (reviewed in [+ cytotoxic T lymphocytes (CTL) recognize conserved internal proteins, like NP and M1 [It has been demonstrated in various animal models, including mice  and nonhiewed in ). BecausP and M1 , 91, theP and M1 . Indeed,P and M1 , 97, H7NP and M1 , H1N1pdmP and M1 , 98, andP and M1 .+ T cells inversely correlated with disease severity in patients infected with the pandemic H1N1 virus of 2009 [+ CTL response was very rapid, which may have contributed to accelerated clearance of the virus [+ T-cell response [+ T cells further support the notion that CTL control influenza virus replication and exert selective pressure on the virus [+ T lymphocytes were also shown to correlate with reduced disease severity [A protective role for cross-reactive virus-specific CTL in humans was first shown after experimental infections . In the  of 2009 , 101. Inhe virus . Furtherresponse . The obshe virus . In a huseverity .The currently available seasonal influenza vaccines  provide Nevertheless, these seasonal vaccines provide little or no protection against zoonotic or pandemic influenza viruses. Thus, upon the emergence of a pandemic, an update of the vaccine with the pandemic virus strain is necessary before the deployment of vaccination programs can occur. This requires swift vaccine development, which is hampered by the current production approach used for the vast majority of seasonal influenza vaccines and based on the use of embryonated chicken eggs. While this technology is relatively efficient and cost-effective, it is associated with a number of major disadvantages. First, generating vaccine candidate viruses by reassortment, and to a lesser extent by reverse genetics, that are highly productive in eggs, is highly time-consuming. The development time of seed viruses, using the backbone of an egg-adapted virus and expressing the HA and NA genes of the circulating viruses, and the subsequent vaccine production time may take as long as 6 to 8 months before the first vaccine doses become available. During this time lapse, new drift variants of seasonal and pandemic influenza viruses alike may arise, resulting in vaccine mismatch. Adaptation of the vaccine seed viruses to replication in avian tissue may also lead to adaptive mutations that may result in yet further mismatch with circulating strains. Such changes may severely reduce vaccine-induced protection . FinallyAlthough the relatively cost-effective egg-based vaccine production platform allows the production of more than a billion vaccine doses annually, several vaccine manufacturers are addressing these shortcomings by the use of accredited cell lines, like African green monkey kidney (Vero) cells, Madin-Darby canine kidney (MDCK) cells, and others as new platforms to produce vaccine viruses at yields that are comparable to those obtained in eggs , 109. SeAmong the seasonal influenza vaccines that are most frequently used today, are the IIVs produced in embryonated chicken eggs. These vaccines have an excellent safety record. Essentially 3 types of IIVs are being used today, based on whole-virions, split-virions, and HA and NA subunits .Classically, alum and oil-in-water emulsions  have been used as adjuvants in some of the nonreplicating seasonal and pandemic human IIVs. These vaccines can profit from the combined use of adjuvants by the resulting increase of the specific immune response upon vaccination, or alternatively by reducing the antigen content in a dose sparing way. The latter may particularly be of interest during an influenza pandemic, when production capacity of vaccine antigen may become a limiting factor for effective vaccination coverage.In addition to adjuvants, high-dose IIVs are produced to increase vaccine immunogenicity. In contrast to the standard dose IIVs (15 µg HA/strain), high-dose IIVs contain 4-fold HA dose. Trials with individuals aged 65 years and older demonstrated a higher antibody response, a better protection against laboratory-confirmed influenza illness, and a reduced hospitalization rate of nursing home residents when high-dose IIVs instead of the standard vaccine were used .+ and CD8+ T-cell responses. To what extent the internal proteins of the cold- and egg-adapted vaccine virus at the origin of CD8+ T-cell responses have been subject to escape mutations over time in the circulating viruses is not clear at present [LAIVs are based on the use of a cold-adapted virus that replicates well in embryonated chicken eggs and better at temperatures lower than the normal human body temperature. These are largely limited to replication in the upper respiratory tract and are therefore attenuated. LAIVs are generated by reassortment and are composed of the internal genes of a cold- and egg-adapted virus combined with the HA and NA of the respective seasonal influenza viruses identified by the influenza vaccine strain selection. As the immunization is based on replicating vaccine virus administered intranasally, it induces a strong local mucosal IgA response. Importantly, it induces both CD4 present , 115.The overall level of protection induced by LAIVs in adults is comparable to that induced by IIVs. However, LAIVs appear to be less effective than IIVs in older adults, while they appear more effective in children. Therefore, the recommended age for this type of vaccine is from 24 months to 49 years of age. The exclusion of children under 2 years of age is related to an increased risk of induction of wheezing. Similarly, because LAIV vaccination depends on replication of the attenuated vaccine virus in the upper respiratory tract, which may result in some mild replication-associated symptoms, certain high-risk groups for influenza and pregnant women also have been excluded.The first purified recombinant HA vaccine FLUBLOK, developed by Protein Sciences and now marketed by Sanofi Pasteur, is formulated into trimer “rosettes,” that are produced in insect cells by a baculovirus expression system. It was shown to be 30% more efficacious than traditional IIVs for adults older than 50 years . This maNext-generation influenza vaccines are urgently needed in order to address seasonal influenza antigenic drift and contribute to better pandemic preparedness , 118. ThSignificant progress towards these next-generation vaccine candidates has been achieved worldwide , 121. VaThe recent discovery of bnAbs against influenza viruses indicates that the generation of a broadly protective vaccine may indeed be attainable. The majority of these bnAbs, however, are directed toward highly conserved conformational epitopes in the HA stem, which lack the immunodominance of epitopes displayed by the influenza HA head. A key strategy proposed to avoid or circumvent influenza HA head immunodominance is by generating recombinant headless HA proteins (or HA stems). However, removal of the transmembrane domain on the one hand and HA head on the other without extensive compensatory modifications to stabilize the remaining molecule leads to loss of native conformation of the HA stem, resulting in low if any presence of conformational bnAb-inducing epitopes. This has led to several approaches currently pursued towards stabilization of HA stems .The HA head is immunodominant, with immune responses naturally targeting the antigenically variable region that surrounds the RBD. However, head-specific bnAbs have also been shown to be induced upon infection. Harnessing HA head immunodominance and steering head-specific immune responses toward more conserved regions of the HA head represents a promising complementary approach. Among explored vaccination strategies to induce reactive antibodies against conserved HA epitopes is the use of sequential vaccination with different chimeric HAs displaying the same HA stem and different HA heads .In addition to HA, several studies have shown that inclusion of NA into influenza vaccines enhances the protective efficacy of these vaccines . Serum a+ T cells in accelerating virus clearance and limiting disease severity upon reinfection with influenza virus is increasingly recognized to potentially play a significant contribution to vaccine efficacy and breadth of protection. The induction of cellular-mediated immunity is largely dependent on replicating viruses and thus, in addition to LAIVs, viral vectored vaccines, as well as DNA- and RNA-based vaccines and virus-like particles are promising new technology platforms towards broader influenza vaccines [+ T helper cell as well as CD8+ effector T-cell immunity, leading to robust and durable protective immune responses. The presentation of viral targets of both humoral and cell-mediated immunity by MVA and other viral vectors has strong potential to optimize and synergize the induction of broad immune responses against influenza.The role of MHC class I restricted CD8vaccines . In partvaccines , 133). Avaccines  and in hvaccines , 138), hvaccines . MVA vecThe striking success of mRNA vaccines against COVID-19 including in high-aged individuals  highlighAdjuvants can improve the vaccine response by enhancing and modulating the immune response. In general, adjuvants act through different mechanisms or a combination thereof. They can create an antigen depot, activate the innate immune response, induce inflammasomes and cytokines, recruit immune cells, improve antigen uptake, enhance immune cell maturation, and change the activation profile of adaptive immune cells (reviewed in ). Up to For instance, multiple studies indicate that the addition of the oil-in-water adjuvant MF59 leads to a faster and higher antibody induction and a better cellular immune repose compared to nonadjuvanted influenza vaccines , 151. MFregs) and myeloid-derived suppressor cells by different kinase inhibitors [regs, an increase in the effector T cells has the potential to lead to an improved ratio favoring immune stimulation [Kinases are one class of biological response modifiers that has been investigated for intervention strategies against influenza viruses. Host kinases not only regulate influenza virus entry and replication, but are also integral components of various antiviral and inflammatory pathways allowing them to shape the immune response (reviewed in  and 158158). Thehibitors . Togethemulation , 166. Soreg inhibition in the skin, suggesting a change of the immune homeostasis in favor of enhanced immune reactions to vaccines [SMKIs have been successfully used as influenza vaccine adjuvants in several murine vaccination/challenge experiments. Topical application of epidermal growth factor receptor inhibitors (EGFRIs) before intradermal vaccination increased the humoral and cellular vaccination response and led to a reduced viral load in the lungs and improved survival rates in challenged mice . Systemivaccines . Anothervaccines . A revervaccines . These rvaccines .Although influenza vaccination is a global achievement and has considerably contributed to reducing morbidity and mortality associated with influenza worldwide, there is an urgent need for novel technologies and strategies to improve influenza vaccine responses towards broader and longer-lasting protective immunity, including in individuals at risk of vaccine failure. The proportion of the population with expected poor vaccine responses is increasing, especially in developed countries. Although there are already strategies to improve vaccine responses for some risk groups, for example the elderly , 169, ad


Liver disease is a global health issue that has caused an economic burden worldwide. Organ transplantation is the only effective therapy for end-stage liver disease; however, it has been hampered by a shortage of donors. Human pluripotent stem cells (hPSCs) have been widely used for studying liver biology and pathology as well as facilitating the development of alternative therapies. hPSCs can differentiate into multiple types of cells, which enables the generation of various models that can be applied to investigate and recapitulate a range of biological activities in vitro. Here, we summarize the recent development of hPSC-derived hepatocytes and their applications in disease modeling, cell therapy, and drug discovery. We also discuss the advantages and limitations of these applications and critical challenges for further development. The liver is one of the most important organs in the body, maintaining homeostasis and performing several other functions, including the elimination of toxic substances, metabolism of drugs and serum proteins, and secretion of bile. The predominant cell types within the adult liver are hepatocytes, cholangiocytes, liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells . Most meRecent advances in human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), have provided promising insight into human biology. They can undergo unlimited self-renewal and differentiate into virtually almost any somatic cell type in vitro. Moreover, the accessibility of patient-derived iPSCs has enabled the successful generation of dozens of disease models for studying disease mechanisms . In receIn recent decades, advances in stem cell biology and an in-depth understanding of liver organogenesis have enabled the development of highly efficient protocols to generate functional HLCs from hPSCs. Most protocols comprise multiple stages that mimic the embryonic development of the liver by activating critical cellular signaling pathways during hepatocyte maturation . TypicalAlthough present protocols have enabled the production of large quantities of functional HLCs derived from hPSCs, the generation of fully mature hepatocytes that are comparable to fresh PHHs, especially in terms of cytochrome P450 (CYP) enzyme induction and drug metabolism, remains difficult. Current HLCs still possess most fetal-like properties, such as the expression of alpha-fetoprotein (AFP), CYP3A (metabolized testosterone), and CYP2D6 (dextrorphan) . MoreoveHLC differentiation efficiency depends not only on culture conditions but also on the quality and origin of stem cell sources. Kajiwara et al. discovered that variations in hepatic differentiation from hiPSCs were mainly caused by donor differences, which might be attributable to the genetic background of the donor cells . When itThe organoid model has proven to be an effective strategy for recapitulating and studying a wide range of biological activities, including organ development, tissue responses to drugs, and disease mechanisms in vitro . This isThe common liver organoids generated in most studies can be classified into epithelial liver organoids and multi-tissue liver organoids based on their cell origin . Typical8 cells, which meets the minimum requirement for human transplant applications ,5450,54]Furthermore, gene-editing technologies, such as CRISPR/Cas9, make it possible to efficiently correct genetic errors in patient-derived hiPSCs c. HLCs dNAFLD encompasses a complex pathologic spectrum from benign hepatic steatosis to non-alcoholic steatohepatitis (NASH), which can ultimately lead to severe cirrhosis and eventual hepatocellular cancer . NAFLD pA more clinically relevant NAFLD model could facilitate the identification of key genes and signaling pathways that regulate disease progression, which could potentially serve as novel pharmaceutical targets. Thus, more efforts should be made to create a microenvironment that enables multi-step disease progression from benign steatosis to severe cirrhosis . To thisHBV and HCV infections are two major risk factors that can cause severe liver cirrhosis and hepatocellular carcinoma (HCC) . EffectiHigh-content screening has been applied to identify novel antivirals using PHHs . NeverthHCC accounts for nearly 80% of primary liver cancers and is often characterized as end-stage liver disease. The complex molecular heterogenicity of HCC makes it almost impossible to establish a standard disease model that recapitulates all disease characteristics . NeverthThe liver is known to exert major metabolic functions upon drugs and toxins absorbed from blood, making it the organ most susceptible to drug toxicity. More than 50% of acute liver failure in clinical settings is caused by drug-induced liver injury (DILI), especially in individuals with pre-existing chronic liver diseases . DILI isDespite recent progress in the use of hiPSC-derived HLCs and liver organoids, the significant caveat resulting from the immaturity of hiPSC-derived hepatic lineages in drug screening should not be overlooked. Indeed, previous comparison studies have reported significant differences in the gene expression patterns associated with drug absorption, distribution, metabolism, and excretion between PHHs and HLCs . One posLiver transplantation is the only curative treatment for end-stage liver disease, yet the shortage of donor organs and the burden of long-term immunosuppression have hindered its wide application. The capability of almost unlimited production of hPSC-derived hepatic cells has made it an invaluable tool as the source for regenerative medicine, disease modeling, and drug development. In particular, patient-derived hiPSCs enable the investigation of individual variance in disease progression and drug responses, which pave the way for the development of personalized medicine. In Japan, hESC-derived hepatocytes were utilized in a clinical trial to examine the safety and efficacy in treating urea cycle disorder , which d


Mycobacterium, Plasmodium, Toxoplasma, and Leishmania infections, which are pathogens that reside within membrane delimited compartments in phagocytes at some time in their life cycle within mammalian hosts. The review includes discussion of the need for further studies into the biogenesis of EVs to better understand the contributions of these vesicles to host-pathogen interactions, and to uncover potential therapeutic targets to control these pathogens.Extracellular vesicles (EVs) have garnered significant interest in recent years due to their contributions to cell-to-cell communication and disease processes. EVs are composed of a complex profile of bioactive molecules, which include lipids, nucleic acids, metabolites, and proteins. Although the biogenesis of EVs released by cells under various normal and abnormal conditions has been well-studied, there is incomplete knowledge about how infection influences EV biogenesis. EVs from infected cells contain specific molecules of both host and pathogen origin that may contribute to pathogenesis and the elicitation of the host immune response. Intracellular pathogens exhibit diverse lifestyles that undoubtedly dictate the mechanisms by which their molecules enter the cell’s exosome biogenesis schemes. We will discuss the current understanding of the mechanisms used during infection to traffic molecules from their vacuolar niche to host EVs by selected intravacuolar pathogens. We initially review general exosome biogenesis schemes and then discuss what is known about EV biogenesis in  There are myriad facets of host–pathogen interactions that contribute to infection outcomes. Such is the case with intracellular pathogenesis, which includes organisms that use the host biosynthetic machinery to propagate themselves, organisms that reside in the cell cytoplasm and organisms that reside within vacuolar compartments in host cells. Progress in our understanding of mechanisms deployed by pathogens to exploit or subvert host mechanisms is uneven. It should therefore be enlightening to consider current knowledge of pathogen-host interactions by pathogens that share important characteristics. Our focus will be on pathogens that reside within membrane delimited vacuolar compartments in the cell. Even among this relatively small subset of pathogens, there is great diversity in pathogen strategies to interface with the cell, including differences in the composition and interactions of the pathogen-containing vacuolar compartments. Each pathogen elaborates unique strategies to translocate molecules from their intravacuolar niche into the cell cytosol or to other host cell organelles where they target host cell processes. It is presumed that some of the molecules that are released into the cell cytosol may access the exosome biogenesis machinery of host cells. The intravacuolar pathogens that we will discuss infect phagocytes at some point in their life cycle. Phagocytes are residents of tissues where they play important roles in tissue homeostasis and disease [reviewed in ]. PhagocNumerous studies have shown that extracellular vesicles (EVs) released by phagocytic cells infected with intracellular pathogens hold infection-specific molecular cargo that may contribute to pathogenesis and host immune responses [reviewed in –4]. Despvia the blebbing of cells undergoing apoptosis  form by inward budding of the early endosome (EE) . Early eThe ESCRT machinery was first shown to play a role in sorting proteins into MVBs in yeast in 2001 . The ESCThe ESCRT subcomplexes perform a series of synchronized tasks to drive both cargo loading and physical membrane-remodeling and scission, leading to the production of ILVs: ESCRT-0 sequesters ubiquitinated cargo, ESCRT-I, -II, and -III control ILV budding, and Vps4 regulates membrane scission . ESCRT-0The PDZ  domain-containing protein syntenin is important for the biogenesis of a specific subclass of CD63-positive ILVs. Syntenin interacts with ALIX, pieces of the ESCRT machinery, and membrane receptors such as syndecans to produce ILVs using an alternative ESCRT pathway of cargo recruitment and vesicle budding , suggestDepletion of all four ESCRT subcomplexes is insufficient to block exosome secretion entirely in eukaryotic cell lines but does result in drastic changes in cell morphology, such as enlarged, empty MVBs and irregularly shaped ILVs . TherefoLipidomic analysis of EVs reveals that exosomes contain an overall low concentration of lipids, resulting in a high protein/lipid ratio . The lipHowever, depletion of ceramide has no effect on MVB biogenesis or exosome secretion in human melanoma cells . In thesExosomal protein composition is selective and dynamic, suggesting that there are regulated mechanisms involved in loading specific target molecules during vesicle biogenesis. Mass spectrometry analysis of exosomes reveals that post-translational modifications (PTMs) such as ubiquitination (mono-ubiquitination and poly-ubiquitination), the addition of small ubiquitin-related modifier (SUMOylation), phosphorylation, and glycosylation are all common modifications of exosome proteins and may be involved in the selective sorting of cargo molecules .via the isopeptide bond of C-terminal glycine of ubiquitin with the ϵ-amino group of a lysine residue present in the target protein, resulting in a complex network of modifications  is a vaccine strain of Mycobacterium. Mycobacteria can infect several cell types, such as neutrophils, macrophages, and monocytes, although alveolar macrophages appear to be the preferred host of Mycobacterium tuberculosis in the early phases of infection  that do not fuse with lysosomes, which ensures that the bacterium survives in the vacuole  appear to be translocated from the vacuole via secretion systems. It has been shown that cell wall constituents of M. avium, glycopeptidolipids, are released by infected macrophages (via Toll-like receptors (TLRs) -2, -4, that are dependent on the MyD88 pathway  of M. tuberculosis induced pathogen‐specific IFN‐γ and IL‐2‐expressing CD4+ and CD8+ T cells. This Th1-biased immune response was specific to mice vaccinated with exosomes obtained from CFP-treated macrophages, while the BCG vaccine boosted the Th2 response. Those exosomes were also shown to prime a protective immune response at a level comparable to BCG and provided a booster to a previous BCG immunization, leading to a decrease in mycobacterial count in lung and spleen of mice challenged with aerosolized M. tuberculosis  fluid from M. tuberculosis-infected BALB/c mice and infected J774 macrophages . This might mean that several different mechanisms, including bacterial lysis, are responsible for the release of mycobacterial proteins prior to their incorporation into exosomes  than uninfected cells. However, transcripts regulating immune response are more abundant in exosomes derived from M. tuberculosis-infected macrophages. Apart from the host RNA, the vesicles are also capable of carrying mycobacterial RNA. Similar to proteins, the RNA cargo is transferred between cells via exosomes , which is in contrast to Toxoplasma whose PVs establish a tight association with the Golgi apparatus, Mitochondria, and Endoplasmic Reticulum (ER)   of the h cytosol . Parasit cytosol . Upon thPlasmodium parasites infect distinct cell types that should be expected to employ mechanisms for exosome biogenesis. Several studies have explored the role of exosomes and microvesicles  in Plasmodium infections. Mature red blood cells are devoid of a nucleus and the endocytic cell machinery in their cytoplasm, including multivesicular bodies (MVBs) that play a critical role in exosome biogenesis in nucleated cells. Plasmodium within mature red blood cells have been shown to translocate over 300 parasite-derived proteins from their PVs into the red blood cell cytosol. These molecules that are distributed throughout the red blood cell, including on its cell surface, have been implicated in several functions, including the formation of Maurer’s clefts that appear to play some role in exosome formation in Plasmodium-infected cells  plays a role in phosphatidylserine distribution at the plasma membrane  that is expressed by a multigene family and has been implicated in virulence , merozoiewed in  describefections . The resfections  in whichlciparum  and P. vP. vivax  that wouexosomes . This anToxoplasma are Apicomplexan parasites that can infect all warm-blooded animals, including mammals and birds. In humans, Toxoplasma have been implicated in a range of clinical presentations whose severity is determined by the individual’s immune status. Toxoplasma is acquired by ingestion of raw or inadequately cooked meat. It can also be acquired upon ingestion of oocyst dispersed in the environment in cat feces. This later mode of acquiring the infection by pregnant women is the subject of public health campaigns that dissuade women from changing cat litter, as infection during pregnancy can lead to congenital transmission resulting in stillbirths or hydrocephalus or retinal infections of the newborn. Toxoplasma can infect all nucleated cells in mammals. Parasite entry into cells involves the sequential deployment of molecules from Apicomplexan-specific organelles. The release of proteins from Rhoptries follows the discharge of micronemal proteins. Dense granule proteins are then released, which contribute to the formation of the parasitophorous vacuole and the intravacuolar network. Rastogi et al.  and Leishmania (Viannia) that are further classified into species and subspecies. Infection of humans results in a disease presentation that is mostly dependent on the parasite species. Parasites of the Leishmania (Leishmania) subgenus including L.(L) donovani, L.(L) infantum and L.(L) chagasi (with a few exceptions including L.(L) amazonensis and L.(L) major, L.(L) tropica that cause cutaneous infections) are the primary causative agents of visceral disease. In contrast, parasites of the Leishmania (Viannia) subgenus, including L.(V) braziliensis, L.(V) panamensis, cause cutaneous infections that can manifest as self-limiting lesions or disseminated lesions or mucocutaneous infections [reviewed in . The Leishmania species determine LPV morphology. At the extremes of morphological differences, parasites of the L. mexicana complex  reside within large communal LPVs that continuously distend. At the other extreme, L. donovani, L. chagasi/L. Infantum reside in tight LPVs that harbor a single parasite. After parasite replication and fission, daughter parasites segregate into secondary LPVs that also house individual parasites. All other Leishmania species reside in LPVs that may house one to four parasites. It is presently not known how LPV morphology differences affect Leishmania pathogenesis. LPVs are dynamic compartments composed of molecules from the host secretory pathway, including the endoplasmic reticulum (ER) and the endocytic pathway, including late endosomes and lysosomes  before dissemination. Recent studies that have explored the phenotype of cells at primary and secondary sites in experimental infections with L. major, highlighted the expression of chemokine receptors CCR2+ and CX3CR1+ on the monocyte-derived cells that are the primary host cells of these parasites  as essential mediators of angiogenesis in L. major infected lesions. Also performing studies with L. major, Weinkopff et al. , also referred to as GP63, is an important virulence factor, which contributes to enhanced phagocytosis by macrophages and promoting the survival of the parasite during both promastigote and amastigote life stages. There are three pools of GP63: surface-localized, internal, or released, that are believed to traffic separately through the cell and then released into the extracellular environment  to evaluate mature infections. After 72 hours of infection, extracellular vesicles were purified from media supernatants using differential centrifugation and then subjected to liquid chromatography combined with tandem mass spectrometry. In-solution and in-gel protocols for tryptic digestion were used prior to the mass spectrometry-based analysis, and data combined to yield protein composition profiles for uninfected and infected macrophage-derived EVs. Consequently, this approach led to a confident identification of 59 parasite proteins in EVs released by infected macrophages. These proteins included a putative Vasohibin, nucleoside transporter 1, kinesin, DNA directed RNA polymerase II subunit 2, dynein heavy chain, and putative protein kinases. Interestingly, some of the same protein families were also identified in a study of circulating immune complexes in the peripheral blood of 115 human patients with active L. donovani infections (Leishmania protein databases currently in use. In addition, it can be challenging to match proteins exactly across different experiments by reported accession numbers. However, the parity between these profiles at the protein family level remains intriguing. Along with what is known about exosomes and their ability to circulate through the body and deliver their contents to tissues distant from their origin, these findings suggest that L. donovani infected macrophages release exosomes containing parasite factors that may aid in their circulation throughout the host.Recent research also indicates that macrophages infected with rocesses . In thatfections . In thatLeishmania promastigote secreted exosomes (Leishmania amastigotes may translocate their molecules across the LPVM into the host cytosol to utilize normal host processes to their advantage for the secretion of specific parasite molecules. EVs released by Leishmania-infected macrophages were also found to enhance several measures of endothelial cell activation in vitro, including tube formation, cell migration, and enhanced production of VEGF and IL8, which suggests that infection-induced EVs may play a role in neovascularization and pathogenesis (It is also worth noting that exosomes released by infected macrophages contain only 59 parasite-derived proteins, a significantly smaller number than the 329 parasite proteins identified in exosomes . Compariogenesis .All eukaryotic cells release EVs, including exosomes. The biogenesis of EVs released by cells under various normal and abnormal conditions has been well-studied. There is ample evidence that intrinsic cell characteristics and environmental queues determine the composition of EVs. This results in unique exosome compositions that could be monitored for diagnostic value. Ongoing studies have shown that intracellular pathogens, including viruses, bacteria, and parasites, can take advantage of the host exosome machinery to release virulence factors. Pathogens may smuggle pathogen-encoded virulence factors and other molecules that collectively contribute to their pathogenesis. The existence of these mechanisms suggests that pathogens have evolved adaptations to take advantage of host protein trafficking mechanisms for exosomal packaging. For its part, the host cells also modulate exosome composition as an anti-pathogen strategy since exosomes induce immune responses directed against specific pathogens. More research is needed to evaluate specific mechanisms by which pathogen-derived molecules are targeted to multivesicular bodies within infected cells and further packaged into host-derived exosomes. This information will elucidate a large gap in our understanding of intracellular host-pathogen interactions and identify novel drug targets for infection control.AG wrote and edited the manuscript. ME wrote and edited the manuscript. PEK developed the manuscript focus, wrote and edited manuscript. All authors contributed to the article and approved the submitted version.PEK was supported by an R56 AI143293 grant from the NIH. ME was supported by the R03 AI-135610 grant from NIH.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Due to the complex structure and function of central nervous system (CNS), human CNS in vitro modeling is still a great challenge. Neurotoxicity testing of environmental chemicals mainly depends on the traditional animal models, which have various limitations such as species differences, expensive and time-consuming. Meanwhile, in vitro two-dimensional (2D) cultured cells or three-dimensional (3D) cultured neurospheres cannot fully simulate complex 3D structure of neural tissues. Recent advancements in neural organoid systems provides excellent models for the testing of environmental chemicals that affect the development of human CNS. Neural organoids derived from hPSCs not only can simulate the process of CNS development, including early stage neural tube formation, neuroepithelium differentiation and regional specification, but also its 3D structure, thus can be used to evaluate the effect of chemicals on differentiation and morphogenesis. Here, we provide a review of recent progress in the methods of culturing neural organoids and their applications in neurotoxicity testing of environmental chemicals. We conclude by highlighting challenge and future directions in neurotoxicity testing based on neural organoids. If fetus exposed to industrial chemicals in the environment, the development of central nervous system (CNS) might be damaged, and thus neurodevelopmental disorders and birth defects will occur, such as attention deficit hyperactivity disorder (ADHD), mental retardation and neural tube defects. There is increasing evidence that exposure to endocrine-disrupting chemicals, particularly phthalates and bisphenol A, may be associated with ADHD , 2. EvenTraditionally, neurotoxicity testing has been based on animal models such as mice, rats, zebrafish and rabbits, which are regularly used to develop human exposure guidelines. By animal models, the researchers preliminarily explored the toxic effects of environmental pollutants on the development of CNS . Ayelet Human pluripotent stem cells (hPSCs), including human embryonic stem cells (ESCs) derived from blastocysts and induced pluripotent stem cells (iPSCs) produced from somatic cells, have the capacity to proliferate extensively and differentiate into multilineages. The two-dimensional (2D) culture model from hPSCs has become an important way to study neurodevelopmental toxicity. However, human CNS tissue is a very complex 3D structure composed of cells and extracellular matrix, 2D cell culture or 3D cultured neurospheres cannot reflect the complete tissue structure. It is impossible to study the effect of chemicals on the structural changes of CNS tissue by 2D cell culture model, therefore, researchers turned to hPSCs derived 3D neural organoids to investigate the neurotoxicity effects of environmental chemicals. Human neural organoids are distinct from traditional neurospheres and 3D tissue engineered constructs consisting of cells seeded on a 3D scaffold. As defined by Lancaster and Knoblic, organoids are 3D cell complex formed by inducing differentiation of hPSCs or organ progenitor cells and self-organizes through cell sorting and spatially restricted lineage commitment in a manner similar to in vivo . InitialIn this review, we analyze the various neural organoids generation protocols from hPSCs and how these models have been used in neurotoxicity testing research. Finally, we discuss challenges for the development of advanced organoid-based in vitro systems for neurotoxicity testing.The development of vertebrate CNS can be divided into three stages: neural induction, patterning formation and neurogenesis. The process of notochord inducing the formation of neural tube is neural induction. Firstly, the dorsal ectoderm is differentiated into neural plate, which is composed of neuroepithelial cells, under the action of neural inductive factors. Then, the edge of neural plate became thicker and uplifted to form nerve fold. Finally, the neural folds fuse in the dorsal midline to form a hollow neural tube, in which the neuroepithelial cells remain undifferentiated, and constantly expand the number of cells through rapid proliferation . EnvironAfter the formation of neural tube, it patterns along anterior posterior (AP) axis and dorsal ventral axis to guide the expression of development related genes in specific cell groups of neural axis. For anterior posterior axis patterning, the traditional view of neural development is that the anterior neural plate is first produced under the action of activation signal. Then, the mesoderm at the relatively late development stage provides “transforming signals”, for example retinoic acid (RA), inducing anterior neural plate to produce midbrain, hindbrain and spinal cord located in the posterior of neural axis. This model is called “activation transformation” model, which suggests that the posterior nervous system, such as the spinal cord, acquires the anterior fate of the neural axis prior to the posterior fate , 18. HowThe neural tube is defined by its anterior posterior axis, but it also has a dorsal-ventral dimension. Dorsal ventral (DV) patterning is essentially a process in which cells located in different positions of DV axis obtain different fate of differentiation. Although it is known that Wnt, BMP from dorsally located epidermis and Shh emanated from the ventrally located notochord are the main morphogen affecting the DV patterning. However, DV regionalization of early embryonic brain is not obvious, and its potential model remains elusive. Recently, researchers propose a model that human forebrain DV patterning also follows an activation/transformation paradigm . They prBrain organoids can be divided in two categories based on their generating methodologies: self-patterning and extrinsically patterning methods . Self-paLancaster and Knoblich firstly created whole-brain organoid or cerebral organoids containing numerous, but discrete brain regions per organoid using a novel spinning bioreactor approach , 29. ThiCurrently, most protocols optimize brain organoid methodologies by applying exogenous cues to mimic endogenous patterning. Yoshiki Sasai group first proposed the method to generate brain organoids with specific region from mESCs. They developed a series of 3D differentiation protocols by the serum-free floating culture of EB-like aggregates with quick re-aggregation (SFEBq) technique , 32. In In the past years, based on the deepening understanding of the mechanism of spinal cord development, researchers have successfully differentiated hPSCs into various types of spinal cord cells, such as spinal motor neurons and differentiation of V2a interneurons , 41. WheTanaka group firstly constructed a 3D neural tube model, called neuroepithelial cysts, which could be patterned to spinal cord organoids in vitro using single mouse embryonic stem cells . This stRecently, Zheng et al. developed a human neural tube model, which could develop into dorsal ventral patterned spinal cord organoid from hPSCs based on a Gel-3D culture system Fig.  46]. Th. Th46]. Anterior-posterior body axis patterning of neural tube is another process that is critical for spinal cord development. Researchers have recognized that the posterior neural tube is partially generated from a unique pool of axial stem cells called NMPs. However, the model of neural tube axial elongation is still lacking, which has prevented understanding specifically how NMPs regulate and coordinate the emergence of the posterior spinal cord in humans. Currently, Libby et al. reported AP patterned spinal organoid model that displayed regionalized rostral-caudal HOX gene expression, with spatially distinct hindbrain (HOXB1+) expression from brachial (HOXC6+) and thoracic (HOXB9+) regions Fig.  47]. Th. Th47]. As described above, spinal cord is formed via sequential morphogenetic processes including polarized neuroepithelial cell induction, sheet-like neural plate formation, and folding-based tube morphogenesis Fig.  48]. Ho. Ho48]. As mentioned above, great progress has been made in the construction of neural organoids. However, due to the limitation of culture technology and incomplete understanding of neural differentiation mechanism in vivo, these organoids still have their own shortcomings. In Table Given the similarity between neural organoid models and early development of CNS, they may be the most suitable models for studying the chemicals that will cause developmental disorders in fetus. With the development of hPSC-based technologies to generate neural organoid, some recent studies have shown potential for neurotoxicity testing assessment of chemicals based on neural organoids. Table Schwartz et al. developed a 3D brain organoid model that provide a faster, cheaper and more biologically relevant way to screen drugs and chemicals that could harm the developing brain . The braXuyu Qian et al.demonstrated the forebrain organoids platform is amenable to chemical compound testing. They tested the effect of Bisphenol A (BPA), which is commonly found in household plastic products and is known to affect rodent neural development . The resPregnant women drinking alcohol during pregnancy can seriously affect the development of fetus brain, leading to a series of symptoms, such as cognitive impairment and psychosis, known as fetal alcohol spectrum disorder, which pathophysiology and mechanism are still unclear. Based on human brain organoids-on-a-chip, Yujuan et al. explored the effects and mechanisms of alcohol exposure on fetal brain development . The braSpina bifida, one type of NTDs, caused by the failure of spinal neural tube closure, which can be a result of environmental factors exposure such as antiepileptic drugs. Ju-Hyun et al. analyzed the effects of 6 antiepileptic drugs with three different concentrations on the NTDs based on spinal cord organoids, which could recapitulate in vitro neurulation-like tube-forming morphogenesis . The resAlthough neural organoids promise to develop next-generation high-throughput screens that can provide more physiologically relevant predictions of neurotoxicity. However, due to the heterogeneity of organoids, it is difficult to distinguish and quantify the data obtained from organoids we wanted. Henrik et al. developed an automated approach for generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates by using robotic technology . The worThe more and more publications of neural organoid-based toxicity testing studies of environmental chemicals demonstrating neural organoids have more extensive prospect. However, these studies utilized different protocols to generate organoid even from the same region. Therefore, defining and standardization the conditions required for the derivation of neural organoid with specific CNS region is critical for their application in toxicity testing. In parallel, combination with engineering technologies such as automation technology, for robust and reproducible generating homogeneous neural organoids at large scale is also important for application of neural organoids in toxicology testing.There are profound species differences in CNS between human and animals, which proves that human neural organoids are superior to animal models. However, like all in vitro models, neural organoids are not identical copies of their in vivo counterparts. Next, we discuss the aspects of neural organoids that are still challenging, which also limit their application in neurotoxicity testing of environmental chemicals, and discuss how current and future advances may help to solve the questions.The lack of blood-brain barrier in neural organoids may have an important effect on understanding the real toxicity and mechanism of chemicals. Many chemicals cross the blood-brain barrier, but some cannot. Although co-culture with vascular endothelial cells may form vascular networks in brain organoid and 3D biomaterial environments, the problems are that vascular-like networks may not penetrate into human brain organoids, the formed networks may not functional, and the existence of blood vessels may interfere with the self-organization of human brain organoids , 65. FutIn addition, the lack of blood vessels limits the transport of oxygen and nutrients in neural organoids, resulting in restricted growth and maturation of organoids. Recently, Stefano Giandomenico and colleagues developed a method for growing a slice of cerebral organoids on a porous supporting membrane such that the tissue is at the air-liquid interface . This alVariability and reproducibility of current neural organoids remains the fundamental limiting factor for organoids’ application in neurotoxicity testing. As mentioned above, region-specific brain organoids generated by patterned protocols showed greater organoid-to-organoid reproducibility both differentiation and cellular composition. However, the method rely on autonomous cell fate patterning in a relatively homogenous culture environment, and thus the efficiency of generating patterned organoids remains relatively low , 68. ThiHigh throughput toxicity screening is a process running millions of environmental chemical tests in a short time to determine their toxic effects. The advantage of high throughput screening is simplicity, rapidness, low cost, non-labor-intensive, high efficiency, as well as leading to a higher information harvest. However, the establishment of high throughput screening approach is accompanied by its own set of challenges. In addition to the above limitations such as vascular deficiency and heterogeneity, the current neural organoid technology still faced with the shortcomings of manual and less intelligent, which makes the efficient, unbiased, high throughput evaluation of neurotoxicants effects by organoid systems challenging. Automation and standardization of cell seeding, maintenance and fixation of organoids can reduce manual intervention and maintain culture process consistency, enabling homogeneous and reproducible neural organoids generation for screening applications. Moreover, Technological advances in high content imaging will be undoubtedly favorable to the application of neural organoids in neurotoxicity screening. Therefore, the establishment of automation neural organoids culture platforms by advanced engineering and robotic technologies will promote organoid’s utility in high throughput screening of neurotoxicants.In summary, various studies have shown that neural organoids can recapitulate some key features of the human brain and spinal cord, including cellular distribution and organization, physiological structure, and neuronal networks. Therefore, neural organoids have become a unique model to evaluate the effects of acute and chronic toxin exposure. However, we should recognize the differences between neural organoids and tissues in vivo, such as the lack of blood-brain barrier and spatiotemporal morphogen gradient. The advances for high-fidelity organoids-generating techniques are driving application of neural organoids in neurotoxicity testing.


Multiple sclerosis (MS) is one of the leading causes of disability in young adults. The onset of MS during developmental age makes pediatric patients particularly susceptible to cognitive impairment, resulting from both disease-related damage and failure of age-expected brain growth. Despite different test batteries and definitions, cognitive impairment has been consistently reported in approximately one-third of pediatric patients with MS. However, the lack of a uniform definition of cognitive impairment and the adoption of different test batteries have led to divergent results in terms of cognitive domains more frequently affected across the cohorts explored. This heterogeneity has hampered large international collaborative studies. Moreover, research aimed at the identification of risk factors  or protective factors  for cognitive decline is still scanty. Mood disorders, such as depression and anxiety, can be detected in these patients alongside cognitive decline or in isolation, and can negatively affect quality of life scores as well as academic performances. By using MRI, cognitive impairment was attributed to damage to specific brain compartments as well as to abnormal network activation patterns. However, multimodal MRI studies are still needed in order to assess the contribution of each MRI metric to cognitive impairment. Importantly, longitudinal studies have recently demonstrated failure of age-expected brain growth and of white matter (WM) and gray matter (GM) maturation plays a relevant role in determining cognitive dysfunction, in addition to MS-related direct damage. Whether these growth retardations might result in specific cognitive profiles according to the age at disease onset has not been studied, yet. A better characterization of cognitive profiles in pediatric MS patients, as well as the definition of neuroanatomical substrates of cognitive impairment and their longitudinal evolution are needed to develop efficient therapeutic strategies against cognitive impairment in this patient population. Multiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). Approximately 2–5% of patients with MS experience their first demyelinating attack prior to age 18 . DespiteNonetheless, the onset of MS during a period of life in which myelination is ongoing in the developing CNS and the child is acquiring cognitive competencies, can have dramatic consequences on neuropsychological functioning . The potAn understanding of the neuroanatomical basis of cognitive impairment in pediatric MS patients is a necessary prerequisite to develop efficient therapeutic strategies against cognitive decline. MRI has proven to be a powerful tool to investigate the neuroanatomical basis of cognitive impairment in adult MS patients. Through its application to pediatric patients, several potential substrates were also individuated in this population.In this narrative Review, we aim to provide a comprehensive and up-to-date perspective of cognitive impairment in pediatric MS patients. We researched the main international databases for research articles published in international peer-reviewed journals and based on a minimum cohort of pediatric MS patients arbitrarily settled as >10 subjects. We selected the following keywords for the research: “multiple sclerosis” and “cognitive”, and “MRI, and “pediatric”, “paediatric” or “children”. Based on our research and expertise in the field, we are going to discuss the clinical correlates and the impact on everyday life of cognitive and mood-related difficulties in pediatric MS patients, as well as their long-term consequences. Moreover, we are going to analyze the role of MRI for understanding of the neuroanatomical basis of cognitive dysfunction in pediatric MS patients, summarizing the main advancements separately for each MRI metric and technique. Finally, we are going to discuss unmet needs and to provide some guidance for future research in the field.Cognitive assessment of pediatric MS patients can be challenging due to the existence of different age- and sex-related developmental trajectories, which require comparison with different normative groups. Moreover, there is no specific consensus on which neuropsychological tests to use, and on what are the best tools for evaluating the functional impact of cognitive issues on subject’s social and academic functioning ,8.Brief cognitive batteries are increasingly used, nowadays, as they have proven to be reliable in detecting cognitive impairment in the pediatric population. Some of these batteries have been specifically developed for pediatric MS patients, such as the Brief Neuropsychological Battery for Children (BNBC) . The BNBAnother brief assessment tool, the MS Inventory for Cognition in Adolescents (MUSICADO) , was recThe SDMT is a reliable index to detect cognitive impairment in adult MS patients. Indeed, it is now recommended as a cognitive screening test in adult MS patients, alone or together with the Rey Auditory Verbal Learning Test (RAVLT) and the Brief Visuospatial Memory Test-Revised (BVMTR) . Since iOther studies adopted the Brief International Cognitive Assessment for MS (BICAMS) ,14 and tMore recently, normative values for the BRB have been published in an Italian adolescent population . This baInformation on the prevalence and the profiles of cognitive impairment in pediatric MS patients is derived from several cohort studies, often including small samples and adopting different test batteries and definitions of cognitive impairment . DespiteIn an Italian multicenter study , cognitiIn a large sample from the US network of MS centers , cognitiInformation about the long-term evolution of cognitive functioning in pediatric MS is still scanty . HeterogIn an Italian cohort , 56 patiForty-eight subjects, belonging to this same cohort were re-assessed after 5 years , showingOn the other hand, in a US-based study  includinAnother relevant aspect to consider is the potential impact of MS onset during childhood on cognitive and functional attainments later in adulthood. An Italian cross-sectional multicenter study compared cognitive performance in adulthood between a group of 119 pediatric-onset MS and 712 adult-onset MS patients . While aOn the whole, while cognitive deterioration appears to be relatively frequent in pediatric MS, many patients may exhibit stable or improved cognitive functioning over time. In this perspective, according to recent recommendations on cognitive assessment in MS .There is little information on clinical correlates of cognitive impairment in pediatric MS. In cross-sectional studies, a worse cognitive performance was mainly associated with younger age at onset ,24, longThe available evidence about potential predictors of cognitive decline over time is even more limited and sometimes conflicting in pediatric MS. Younger age at disease onset was associated with a higher likelihood of cognitive decline on measures of IPS ,33,34 anIn summary, there is a dearth of studies on predictors of cognitive decline over time in POMS patients, with conflicting and insufficient evidence about age at onset and sex as potential predicting factors.A number of potential protective factors against cognitive decline have been suggested. In an Italian 5-year longitudinal study, higher IQ scores were associated with stable or improving cognitive performance at subsequent evaluations, particularly in cognitively-preserved subjects at the first assessment . Positivp = 0.03) [n = 4850), pediatric-onset MS was associated with less educational achievement, lower earnings, and greater use of disability benefits in adulthood [The occurrence of cognitive impairment in pediatric MS patients can have a significant functional impact. In the US-based study , 35.1% o = 0.03) . Lower e = 0.03) . Likewisdulthood .Depression is present in up to 40% of adult MS patients ,41,42.In general, parents are more prone to report mood disorders in their children as compared with patients’ self-report . For insMood disorders in pediatric-onset MS patients are not without consequences. Depression and anxiety can negatively affect quality of life scores along with fatigue, disability, and disease duration ,46. MoreIn light of above evidence, systematic evaluation of mood disturbances, together with cognitive abilities, is of critical importance in order to provide prompt and appropriate interventions.MRI plays a pivotal role in the diagnosis of MS, in monitoring disease progression and treatment response, so much in children as it does in adults. In details, current goals of MRI in pediatric MS include: confirmation of a diagnosis of MS before a second clinical attack  in indivMRI scans of pediatric MS are typically characterized by multiple white matter (WM) lesions. In adolescents, the characteristics of these lesions are similar to those reported in adults: frequently located asymmetrically in the periventricular and juxtacortical WM regions, the corpus callosum and infratentorial regions, and characterized by oval or elliptical shapes . To the However, a preferential involvement of infratentorial regions by lesions was described in pediatric MS, with pontine lesions being particularly prominent in male patients ,70. ThesHigher lesion volumes were associated with cognitive impairment in pediatric MS patients . MoreoveIn addition to focal WM lesions, regions of normal appearing white matter (NAWM) often contain abnormalities in MS, including axonal spheroids and swellings, mild inflammation, microglial activation, gliosis, and increased expression of proteolytic enzymes . These aDiffusion tensor imaging (DTI) represents a powerful tool to assess NAWM microstructural integrity. By using this MRI technique, significant associations were found between NAWM abnormalities and cognitive functioning in pediatric MS patients. Till and colleagues  exploredin vivo [By suppressing the signal from both WM and cerebrospinal fluid (CSF), double inversion recovery (DIR) sequences provide a good lesion contrast in the grey matter (GM), thus improving the detection of cortical lesions in vivo . Comparein vivo . Neverthin vivo  investigin vivo . These rin vivo  in a groin vivo  in a groin vivo  of thesein vivo ,80. Fronin vivo . MS-related GM atrophy develops following a trajectory that starts from the deep GM nuclei and spreads, over time, to many cortical GM regions ,82. In tTill et al.  indicateAnother determinant structure for cognition in pediatric MS was found to be the amygdala, whose lower volume was associated with a lower level of competency in functional communication skills . The anaRecently, Fuentes et al.  applied The hippocampus is known to play a major role in cognitive processes. However, only one study  investigMoving to the cortex, atrophy of the precuneus was related with reduced cognitive performance in pediatric MS patients . Indeed,Finally, the cerebellum represents a strategic node in various segregated networks , showing multiple connections to and from different cortical areas of the forebrain, the thalamus, and the spinal cord. Within this framework, it is not surprising that cerebellar posterior lobe volume reduction can adversely impact cognitive function and especially information processing speed and vocabulary abilities in pediatric MS patients , which iThe poor correlation between structural damage and clinical phenotype might be explained by interindividual differences in brain capacity to respond to damage, in terms of both: recovery from tissue damage (remyelination)  and cortConsidering the difficulty to perform task-dependent studies in the pediatric population, only few studies adopting this paradigm have been conducted to date. Starting from the consideration that abnormalities in sustained attention are frequently associated with behavioral, learning, emotional, and cognitive difficulties in adolescence, and that attention is one of the most frequent areas of cognitive impairment in pediatric MS patients, De Meo et al. , investiIn details, sustained attention system activity was studied with fMRI during the Conners Continuous Performance Test (CCPT), and the structural integrity of the connections between brain regions relevant to the task was measured by using DTI. Paralleling the findings of volumetric analysis , the cenAnother study conducted in cognitively preserved pediatric-onset MS aimed to assess the patterns of fMRI activity during the Alphaspan task , a workiContrary to task-dependent fMRI, multiple RS fMRI studies were performed. The analysis of RS functional connectivity (FC) within brain networks disclosed that functional abnormalities of precuneus paralleled abnormalities detected by structural MRI in pediatric patients with cognitive impairment ,64. MoreSimilar to structural imaging, the cerebellum is another region whose RS FC plays a major role in determining cognitive functioning in pediatric MS patients. Indeed, compared to both HC and cognitively preserved pediatric MS patients, cognitively impaired patients showed a widespread reduction of RS FC, not only between the dentate nucleus and the basal ganglia, but also between the dentate nucleus and bilateral regions located in the parietal, frontal and temporal lobes .Across differing test batteries and definitions of cognitive impairment, cognitive impairment is consistently reported in approximately one-third of pediatric patients with MS. It can have important negative consequences on everyday functioning and school performance as well as social and professional attainment in adulthood.An early and systematic neuropsychological assessment is recommended in pediatric-onset MS for appropriate counselling and management. A prompt identification of subtle cognitive changes should promote the introduction of effective pharmacological and rehabilitative interventions, taking advantage of brain plasticity and compensatory abilities that are deemed to be more pronounced in this age range. In this regard, the assessment and potentiation of cognitive reserve through intellectual enrichment appears to be particularly relevant and promising. On the other hand, as for adult MS patients, cognitive measures should be included among clinical outcomes in trials evaluating the response to treatments.Moreover, further studies focusing on relationships between cognitive functioning and brain MRI abnormalities are needed, especially with multimodal and longitudinal designs, which are bound to shed light on underlying pathophysiology. Despite several substrates of cognitive dysfunction have already been identified by MRI, further work is necessary from a translational point of view in order to establish a prognostic and monitoring value of MRI metrics in the field of MS-related cognitive impairment.Longitudinal studies, including neuropsychological and multimodal MRI assessment, are mandatory in the pediatric MS population in order to unravel the complex interplay between brain development, MS-related brain damage and subsequent adaptive and maladaptive phenomena. A better characterization of cognitive impairment and of the underlying pathophysiological mechanisms might help individuate targets for novel pharmacological and rehabilitative treatment approaches, bringing hope for the future of this population.


Age-related macular degeneration (AMD) affects various aspects of visual function compromising patients’ functional vision and quality of life. Compared to visual acuity, contrast sensitivity correlates better with vision-related quality of life and subjectively perceived visual impairment. It may also be affected earlier in the course of AMD than visual acuity. However, lengthy testing times, coarse sampling and resolution, and poor test–retest reliability of the existing contrast testing methods have limited its widespread adoption into routine clinical practice. Using active learning principles, the qCSF can efficiently measure contrast sensitivity across multiple spatial frequencies with both high sensitivity in detecting subtle changes in visual function and robust test–retest reliability, emerging as a promising visual function endpoint in AMD both in clinical practice and future clinical trials. Age-related macular degeneration (AMD) is currently the leading cause of visual impairment in subjects older than 50 years of age in developed countries and the third-leading cause worldwide . Given tThe current gold standard for the evaluation of visual function in the clinical practice dates back to 1862, when Herman Snellen developed a standard letter chart that has widely remained in use with only minor modifications ever since. By showing progressively smaller letters at a given distance and at high contrast, the Snellen chart assesses the minimal angle of resolution  which defines visual acuity, the visual system’s resolving power. Similarly, the Early Treatment Diabetic Retinopathy Study (ETDRS) chart measures the minimal angle of resolution to define VA, but in contrast to the Snellen chart, it uses a regular geometric progression of the size and spacing of the presented letters, employing a logarithmic scale with increments of 0.1 log units. The scoring method for both is the line assignment method, where a patient gets credit for lines, not letters read. Partially read lines are given a ‘ + ’ or ‘ − ’ designation, though there is no accepted standard on how this is recorded.Although VA is the traditionally used functional outcome metric both in the routine clinical setting and in clinical trials, it seems that it may not be the ideal test, especially in AMD. VA is a relatively crude test that fails to reveal subtle changes in visual function  . FurtherFurther, VA is tested with the ETDRS chart in clinical trials versus the Snellen chart in clinical practice, with translational discrepancies being inherently linked to how VA is currently tested. A recent study looking at VA differences between the two widely used charts in subjects entering clinical trials, reported a significant VA variability of 0.14 logMAR (6.7 letters) in both non-neovascular (dry) and neovascular (wet) AMD and progressively increased VA variability was observed with progressively worse VA .Despite the well-known limitations of using VA to characterize early functional deficits or quantify disease progression, VA remains the most widely accepted and used metric in evaluating visual function in AMD. To better assess visual function in AMD, various alternative metrics of functional outcome have been investigated, including dark adaptation , micropePerhaps the most important pre-requisite of any functional outcome metric is its patient-relevance. This can be assessed by the correlation of the functional metric with functional vision, which is approximated by vision-related quality of life (VRQoL) questionnaires ,34. PondFurther, compared to VA, contrast sensitivity might be affected earlier in the course of neurodegenerative ocular disorders, which allows for earlier detection of deficits in visual function before the effect on VA has become apparent ,29,30. IA functional outcome metric more sensitive than VA to detect subtle changes in visual function following treatment would also be able to detect smaller differences between different treatment arms, which in turn would mean that smaller sample sizes would be required for clinical trials to be adequately powered. Contrast sensitivity has shown promise for measuring the progression of vision loss in eye diseases or their remediation with treatment. Importantly, contrast sensitivity has been able to detect anti-VEGF treatment effects on visual function in cases where VA improvement seems to have stalled ,32. BansDespite its promising role in visual function assessment, the practical constraints limiting the sensitivity and/or precision of most contrast sensitivity testing methods have thus far prevented wider adoption of contrast sensitivity function in clinical practice and clinical trials.Conventional laboratory testing can measure contrast sensitivity function over an expansive range of spatial frequencies and contrast levels combinations . Yet, unDue to severe testing time constraints, clinical contrast sensitivity tests are far more condensed (and less precise) than laboratory testing. As early as 1977, Sjostrand and Frisen used sinusoidal gratings of variable contrasts and spatial frequencies on a television-based display to test patients with macular disease versus controls . Since tFirst introduced in 1988, the Pelli-Robson contrast sensitivity letter chart is the most widely used test to measure contrast sensitivity . All letAs in most retinal disorders, the Pelli-Robson is the most commonly employed contrast sensitivity test in AMD studies. In the non-neovascular (dry) form, Roh et al. and Ponderfer et al. used the Pelli-Robson chart to measure contrast sensitivity and reported associations with quality of life in AMD patients ,35. TreaIn order to measure contrast sensitivity in different spatial frequencies, several tests have been introduced, yet for portability and ease of application these tests are pre-printed letter charts with pre-determined, hence a priori limited, frequencies and contrast levels .The Vistech Vision Contrast Test System test was first introduced by Ginsburg in 1984 . The tesKleiner et al. applied Vistech testing in a small cohort of dry AMD patients, demonstrating a loss of contrast sensitivity at high spatial frequencies relative to normal controls . The CSVThe population-based Blue Mountains Eye Study used the CSV-1000 chart to measure contrast sensitivity yet there were no normal values for reference available and the authors mention that they used this test because it was the ‘simplest test available at the time’ and because ‘there were no practical, well-validated alternatives’ .The FuncAll these pre-printed grating charts measure contrast sensitivity in different spatial frequencies and do not require the patient to have knowledge of the Roman alphabet. Yet, repeated studies have shown these charts have poor test–retest reliability ,50,51,52The Spaeth/Richman Contrast Sensitivity Test (SPARCS) is a computer-based test that measures contrast sensitivity thresholds in a patient’s central and peripheral vision. Patients are asked to fixate on the central area of a grid with nine boxes while the peripheral boxes show various vertical square-wave gratings. Patients are asked to identify which area showed the vertical square wave gratings. A contrast threshold is measured for each of the tested areas that correspond to a supero-temporal, inferotemporal, inferonasal, inferotemporal, and central vision field for each eye .Overall In 2010, Lesmes et al. introduced the quantitative contrast sensitivity function (qCSF) method . This noThe qCSF estimates contrast sensitivity function by presenting spatially filtered optotypes to the patient that modulate in both spatial frequency and contrast, thus enabling the efficient testing of contrast sensitivity across multiple spatial frequencies in parallel . The qCSUnlike older contrast sensitivity tests like the Pelli-Robson chart  that use2 and resolution of 1920 × 1080 pixels, and a tablet held by the examiner.  that maximizes the expected information gain  in a wayUsing a novel active learning algorithm, the qCSF estimates the two-dimensional curve of the contrast sensitivity function, which describes thresholds as a function of spatial frequency. Only 25 trials are needed to estimate the broad, global metric of contrast sensitivity represented by the area under the logarithm of contrast sensitivity function (AULCSF), which represents CSF estimates integrated across spatial frequencies ranging from 1.5 cycle per degree (cpd) to 18 cpd  Figure . Other oThe qCSF method has been used in preliminary studies to measure CSF in several clinical populations including amblyopia ,70 multiOur team initially investigated the clinical utility of qCSF in a cohort of eyes with retinal vein occlusion (RVO). Relative to age-matched controls, eyes with RVO demonstrated statistically significant reductions in all outputs of the qCSF, including AULCSF, contrast acuity, and contrast sensitivity thresholds at all spatial frequencies . In the Our team has also previously reported on the qCSF method in eyes with macula-off rhegmatogenous retinal detachment (RRD) after surgical repair. Eyes with a history of macula-off RRD had statistically significant reductions in AULCSF and in contrast sensitivity thresholds across all spatial frequencies compared to healthy age-matched control eyes . Similar−1, compared to 93 healthy control eyes. In our recently published work , our teaPreliminary unpublished data on a large cohort of dry AMD patients at our institution suggests that CSF as measured by the qCSF is significantly decreased in early dry AMD compared to healthy controls, despite no differences in VA . FurtherThe currently ongoing MACUSTAR Consortium seeks to investigate multiple functional, structural, and patient-reported potential endpoints in intermediate AMD . ContrasIn the field of neovascular (wet) AMD, the use of contrast sensitivity function as a more sensitive functional outcome may demonstrate larger treatment effects after therapeutic interventions such as anti-VEGF injections compared to VA, larger difference in the treatment effect of different therapeutic interventions and even treatment effect when VA improvement may have plateaued. Current trials for combination therapies for neovascular (wet) AMD face the challenge of measuring the superiority of a combination effect, relative to monotherapies that provide a strong comparator signal with high efficacy. There is a critical unmet need for a visual function endpoint that can provide the sensitive and precise signals required to initiate and track neovascular AMD treatment over time. Such an endpoint would provide the valuable data about neovascular AMD therapies needed by regulators to determine their benefit-risk profile and by payors to evaluate their cost-effectiveness.Quantifying meaningful changes in vision is constrained by what is clinically measurable at baseline, during disease progression, or after therapeutic intervention. As researchers work to develop new treatments for both non-neovascular and neovascular AMD, employment of better visual function metrics would aid in detecting subtle changes in visual function and evaluate disease progression and the effectiveness of novel therapeutics ,72,83.Compared to visual acuity, contrast sensitivity seems to correlate better with subjective visual impairment and vision-related quality of life, may be affected earlier in the course of neurodegenerative disorders ,29,30 an


Invasive fungal infections (IFI) are a common infection-related cause of death in immunocompromised patients. Approximately 10 million people are at risk of developing invasive aspergillosis annually. Detailed study of the pharmacokinetics (PK) and pharmacodynamics (PD) of antifungal drugs has resulted in a better understanding of optimal regimens for populations, drug exposure targets for therapeutic drug monitoring, and establishing in vitro susceptibility breakpoints. Importantly, however, each is an example of a “one size fits all strategy”, where complex systems are reduced to a singularity that ensures antifungal therapy is administered safely and effectively at the level of a population. Clearly, such a notion serves most patients adequately but is completely counter to the covenant at the centre of the clinician–patient relationship, where each patient should know whether they are well-positioned to maximally benefit from an antifungal drug. This review discusses the current therapy of fungal infections and areas of future research to maximise the effectiveness of antifungal therapy at an individual level. Aspergillus spp., multidrug resistant Candida auris, and the emergence of new pathogens . Furthermore, invasive fungal diseases are increasingly seen in non-classical settings, such as critically ill patients, and as a complication of influenza, SARS-CoV-1 and SARS-CoV-2 [Invasive fungal infections (IFI) are a common infection-related cause of death in immunocompromised patients. Even with the application of state-of-the-art diagnostic testing and deployment of modern antifungal therapy, the mortality of IFIs remains high . MortaliRS-CoV-2 . Candida spp. and Aspergillus spp. are also reasonably well-characterised. This knowledge has resulted in a better understanding of desired regimens for populations, drug exposure targets for therapeutic drug monitoring (TDM), and establishing in vitro susceptibility breakpoints. Importantly, however, each is an example of a “one size fits all strategy”, where complex systems are reduced to a singularity that ensures antifungal therapy is administered safely and effectively at the level of a population. Clearly, such a notion serves most patients adequately but is completely counter to notions of precision therapy at an individual patient level. The clinical pharmacokinetics of antifungal agents are generally well-characterised . The phaThis viewpoint discusses the current therapy of fungal infections and areas of future research to maximise the effectiveness of antifungal therapy at an individual level. Reviews and guidelines of antifungal drug therapeutic drug monitoring are published elsewhere ,21,22,23Clinical studies used for licensure involve a relatively homogeneous population—a cohort of patients that fulfil typically restrictive inclusion criteria. Patients with suboptimal outcomes that may benefit from a precision approach may only be apparent from post hoc analyses  or from subsequent case reports and/or cohort studies obtained post-licensure. While such evidence is often biased, it helps identify clinical scenarios where a fixed dosing strategy may be inadequate and tailored, or an individualised approach is more appropriate. The following summarises the microbiological, pharmacological and clinical features where precision approaches may be considered.Candida and most Aspergillus species, but there are important exceptions. For example, a group of Aspergillus spp.—A. lentulus, A. udagawae, A. viridinutans and A. fischeri—are less susceptible to first-line anti-Aspergillus agents , thus limiting standard therapeutic choices [The appropriate therapeutic choices and the probability of therapeutic success are dependent on the causative fungal pathogen. The overall therapeutic response to antifungal therapy is generally comparable for different species of  choices . Candida parapsilosis; fluconazole versus Candida glabrata), echinocandins  and polyenes  [Some fungal species display intrinsically reduced susceptibility to licensed antifungal agents. These include species less susceptible to triazoles  . In thesIn vitro susceptibility testing enables further refinement of therapeutic choices and decisions as to whether regimen intensification is a viable strategy. This should be performed by specialised laboratories using standardised techniques with results interpreted according to breakpoints published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)  or the CCandida, Aspergillus, and Cryptococcus are well-defined and can guide the likelihood of success with regimen intensification. A higher MIC requires a proportionally higher drug exposure to achieve a comparable therapeutic response. With regard to this, recently, EUCAST  has re-defined the intermittent susceptibility class to signal regimen intensification, which is required to secure a favourable clinical outcome [The effect of the relevant underlying resistance mechanisms  is generally well-captured by the MIC. PK/PD targets for most of the triazoles against  outcome . The MICThe success of therapy is dependent on the sites of infection. Examples of fungal diseases that are associated with poor clinical outcomes include infections within the central nervous system, endovascular infections, and disseminated disease . Poor ouAspergillus nodules in the lung), and disseminated disease is probably significantly underdiagnosed. A high fungal burden may increase the probability of a resistant subpopulation being present at the time of treatment initiation and progressively expanding [A high fungal burden may also be an important determinant of therapeutic response. Multifocal disease in a single organ is common  can suggest risk for fungal infection and potentially help stratify patients into high- and low-risk groups [There are many factors that are predisposed to invasive fungal diseases. Significant systemic immunocompromise is generally a prerequisite for development of disease  ,46. Incrcii, etc . With thk groups .The main implication of the immunological deficit for the delivery of precision therapy is an objective assessment of the degree of underlying immunosuppression. Regimen intensification  may be required to affect a therapeutic response in a profoundly immunosuppressed host.The pharmacokinetics of antifungal drugs are typically highly variable because of absorption issues , variation in protein binding (resulting in changes in free concentrations), drug–drug interactions and genetic polymorphisms in oxidative metabolism, errors in administration , and inFinally, variability in drug exposure may result from poor compliance and a reduced amount of active substance within counterfeit drugs . A thoroThis is perhaps the most common clinical scenario. Most patients in this group have significant underlying immunocompromise and possible invasive fungal infection (as defined by EORTC/MSG diagnostic criteria) . Fungal In the absence of any microbiological data, there is no alternative but to use TDM drug exposure targets that have been defined for populations . If therThe assessment of the clinical response is notoriously difficult and requires experienced clinical judgement. Clinical signs and symptoms are generally nonspecific and affected (confounded) by multiple comorbidities. Nonspecific biomarkers  may be useful as well as specific biomarkers, such as galactomannan . RadioloIn this scenario, the invading pathogen has been cultured, and an MIC is available. This is commonly seen in candidemia, endemic fungal infections, and cryptococcal meningitis, but remains frustratingly low for mould infections, such as invasive aspergillosis, and infections caused by Mucorales ,83. For Candida albicans, where an AUC/MIC of 100 is a well-cited and commonly accepted drug exposure target [min/MIC target of 2–5 has been estimated from a large number of fungal infections treated with voriconazole [Standard TDM based on PK–PD targets can be used  but are conazole  and is uCryptococcus and Taloromyces in CSF and blood can be used to follow the treatment response of cryptococcal meningitis and talaromycosis, respectively [Medical mycology has multiple examples where the response to antifungal therapy can be quantified in real-time using a variety of fungal biomarkers. Galactomannan can be used to follow the course of invasive aspergillosis . Beta-glucan has prognostic value for invasive aspergillosis and invasive candidiasis. Finally, quantitative fungal cultures of ectively ,85. CleaThe trajectory of a fungal-specific biomarker may be affected by both the organism and/or host-specific factors (An integrated approach addresses “true individualised therapy” or truly “precise therapy”. Each patient has their own individual drug exposure target, which is formed from a combination of host- and pathogen-specific factors. The use of both pharmacokinetic and pharmacodynamic data enables patients to understand whether they are well-positioned to maximally benefit from an antifungal drug and guide treatment decisions tailored for that individual.With the increasing number of immunocompromised patients in the world, treatment of fungal infections will become progressively important. Here, we have presented the current approach to the diagnostics and optimisation of therapy and potential future directions. Whole genome sequencing, biomarkers, disease progression modelling, and FDG-PET/CT may all have an important role in the precision dosing of antifungal agents.