Glioblastoma (GBM) is one of the greatest challenges facing neuro-oncology today. Current treatments are far from satisfactory and, given the poor prognosis of the disease, therapeutic efforts are focused on palliative management rather than curative intervention. Here, we review the cellular heterogeneity of GBM, including tumor cells and microglia/macrophages among others, as well as the genetic, epigenetic and metabolic alterations controlling its initiation and progression. Then, we describe the genetic and xenotransplantation zebrafish models established in the last few years for the study of GBM physiopathology and for testing new drugs to improve the treatment of the disease. Taking this information into account, forthcoming studies using zebrafish models of GBM are expected to shed light on better diagnosis and treatments, thus providing hope for GBM patients.Glioblastoma (GBM) is the most common of all brain malignant tumors; it displays a median survival of 14.6 months with current complete standard treatment. High heterogeneity, aggressive and invasive behavior, the impossibility of completing tumor resection, limitations for drug administration and therapeutic resistance to current treatments are the main problems presented by this pathology. In recent years, our knowledge of GBM physiopathology has advanced significantly, generating relevant information on the cellular heterogeneity of GBM tumors, including cancer and immune cells such as macrophages/microglia, genetic, epigenetic and metabolic alterations, comprising changes in miRNA expression. In this scenario, the zebrafish has arisen as a promising animal model to progress further due to its unique characteristics, such as transparency, ease of genetic manipulation, ethical and economic advantages and also conservation of the major brain regions and blood–brain–barrier (BBB) which are similar to a human structure. A few papers described in this review, using genetic and xenotransplantation zebrafish models have been used to study GBM as well as to test the anti-tumoral efficacy of new drugs, their ability to interact with target cells, modulate the tumor microenvironment, cross the BBB and/or their toxicity. Prospective studies following these lines of research may lead to a better diagnosis, prognosis and treatment of patients with GBM. Glioblastoma (GBM) is one of the greatest challenges facing neuro-oncology today. This disease represents the most common of all brain malignant tumors, comprising more than 50% of existing high-grade gliomas. GBM has an annual global incidence of 3–5 cases per 100,000 inhabitants and a slight predominance in males. This incurable malignant tumor, with almost non-existent long-term survivors, can occur at any age but presents a clear peak with the highest incidence in the sixth decade of life .Most GBM arise de novo, while secondary GBM tumors commonly develop from lower grade gliomas . The comIn this scenario, different promising lines of research have been initiated for the development of new therapeutic strategies to treat GBM . From a Danio rerio) has become a well-established model for studying the physiopathological features and screening of new treatments for several human diseases, including cancer. Rapid embryo development, its small size and its transparency, genetic and physiological conservation and ethical and economic advantages have made zebrafish stand out from all other in vivo models [The zebrafish  manipulation. Then, we provide our view on the opportunities and challenges of zebrafish models for improving our understanding of the disease and the evaluation of new treatments.It is desirable to understand the cellular origin of the disease and the composition of the TME to establish an early diagnosis, identify therapeutic targets and improve its outcome. The genomic, epigenomic and transcriptomic characterization of GBMs has provided information which allowed to establish the different tumor subtypes: proneural, mesenchymal and classical ,17,18,19The glioma tissue is infiltrated by many cells of different ontogenies, mostly resident microglia and tumor-infiltrating monocytes, which are denoted glioma-associated macrophages/microglia (GAMs) and which can represent from 30 to 50% of the total cells in the tumor ,25,26. HAlthough the mechanisms by which GAMs migrate to the tumor site are not entirely clear, some studies have shown that their recruitment is mediated by various glioma-derived factors such as CCL2, CX3CL1, SDF-1, CSF-1, GM-CSF and EGF, which act as chemoattractants for GAMs and mediate the crosstalk between tumor cells and the innate immune system  Figure . In an NPhenotypically, it is now well documented that GAMs do not fit with the classical M1/M2 macrophage dichotomy . OriginaIn the context of glioma, using genome-wide microarray analysis, Szulzewsky et al. revealed that a significant amount of genes up-regulated in GAMs do not fit with the M1/M2 classification  and the To date, the vast majority of studies have demonstrated that macrophages/microglia support GBM progression, although, in some cases, the ability of these cells to fight against the tumor has been reported. This work has also revealed some molecular targets offering possibilities for therapeutic intervention. Although, to our knowledge, studies testing macrophage-directed therapies to treat GBM have still not been performed in zebrafish, several investigations have already evaluated the role of macrophages and microglia in zebrafish regeneration, tumor origin and progression ,60 and iThree important genetic events drive the development of GBM: the dysregulation of growth factor signaling , the activation of the phosphatidylinositol-3-OH-kinase (PI(3)K) pathway and the inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways . IDH mutvIII, contains an in-frame deletion within the extracellular domain that provides constitutive activation in a ligand-independent fashion and promotes cell proliferation via the Ras-MAPK and PI3K pathways. Deletion of PTEN displays a similar effect [As an example of the dysregulation of growth signaling commonly occurring in GBM, 57% of GBM shows evidence of gains in function mutation and/or focal amplification of EGFR, associated with an increase in the aggressiveness of these gliomas . The mosr effect .INK4α in chromosome 9p) are linked to the activation of the Rb pathway and to the proliferative niches that are observed in gliomas [Cycle checkpoint proteins are often altered in cancer. p53 is a classic tumor suppressor that regulates many genes involved in the cell cycle and apoptosis cascades. Inactivation of p53 in GBM happens with a variety of mechanisms, including amplification of p53 inhibitors such as murine double minute (MDM) 2 and MDM4 (the latter appears most commonly in tumors with no TP53 or telomerase reverse transcriptase (TERT) mutations), deletion of p53 stabilizers such as p14/ARF and mutation in the TP53 gene which occurs in 85% of GBMs [ gliomas . Other a gliomas ,68.Genetic mutations are not the sole participants in the development and progression of GBM. Epigenetics, as shown in many other types of cancer, plays an important role too. GBM commonly presents an extended genomic epigenetic hypomethylation that allows the transcription of multiple genes and is associated with rapid progression. Some of the regions affected include D4Z4 (a polymorphic repeat structure), oncogenic genomic loci such as SAT2 and the oncogene MAGE-A1 . MutatioA key example that highlights the importance of epigenetics in GBM comes from MGMT alterations, one of the gold standard markers used for the characterization of the disease. The methylation of the MGMT promoter is a biomarker with great diagnostic value that correlates with good prognosis. Furthermore, as with the DNA alkylation repair enzyme, the inhibition of MGMT expression enhances the anti-tumoral efficacy of TMZ .The epigenetic inactivation of many tumor suppressor genes has also been observed in GBM. PTEN, p53, RB, p14 and p16 appear hypermethylated, along with some other genes involved in key processes such as intercellular contact (PCDH-γ A11), the MAPK pathway (SOCS1) and apoptosis (caspase-8) . HistoneMetabolic switching is a well-known hallmark of cancer, including GBM and understanding and reprogramming metabolic pathways in tumors may yield new treatment options . Many caIt is of note, that mitochondria are a key cellular organelle regulating energy metabolism, generation of free radicals and apoptosis. Additional metabolic signs of malignancy in GBM have been linked to mitochondrial dysfunction and morphological abnormalities, which have been correlated with compromised energy/ATP production via OXPHOS and reduced apoptosis. Impaired mitochondrial metabolic capacity in glioma cells has been evident from the identification of mutations in gene coding for IDH, described in In addition, to genetic and epigenetic intervention, a broad variety of biological molecules have been evaluated for the therapeutic manipulation of metabolic pathways, including sugars, lipids and proteins . FurthermiRNAs are small (18–25 nucleotides), evolutionarily conserved, non-coding RNAs with important functions in gene regulation, acting predominantly at the posttranscriptional level. miRNAs are transcribed in the nucleus by RNA polymerase II into primary transcripts (pri-miRNAs) that are then processed sequentially in the nucleus and cytoplasm by a complex of RNase III-endonucleases, namely Drosha and Dicer ,84,85, . Drosha,Over the past two decades, miRNAs have been shown to regulate many physiological processes in the cells and they have been implicated in a wide range of pathological conditions, including cancer. Around 50% of the human-miRNA-encoded genes are located in genetic loci associated with cancer  and nearThe role of different miRNAs in regulating classic pathways and genes critical for the GBM genesis at the post-transcriptional level has been described. These include p53, EGFR, PDGFR, PTEN, PI3K and AKT and MGMT, among others  Figure . MicroRNOverall, this knowledge and further understanding of molecular mechanisms controlling GBM pathology will offer numerous possibilities for therapeutic manipulation of signaling pathways, often intertwined, which can theoretically be treated with different drugs at the genetic, epigenetic or metabolic level. Genetic or xenotransplantation models of zebrafish selectively manipulated in these key pathways are particularly suited to the testing of new therapeutic strategies. In this context, miRNAs represent an attractive target which can be efficiently blocked by (i) sequence-specific oligonucleotides, (ii) antisense approaches or (iii) miRNA sponges.Over the last few decades, advances in biotechnology have allowed improved in vitro and in vivo models of human disease to be developed, underlying pathogenic mechanisms to be understood and therapeutic approaches to be improved. Although in vitro systems for cancer research have experienced significant advances, living organisms bearing tumors are still necessary to recreate the “atmosphere” of human tumors; such examples could be the interaction between tumor cells and immune cells in the primary tumor or at metastatic sites, endocrine or metabolic factors altering tumor development, not to mention biodistribution, pharmacokinetics or efficacy of new drugs ,102. DesThe success of zebrafish in biomedical research can be explained by its inherent biological features and economic issues. Zebrafish are able to produce between 200 and 400 embryos per couple once a week and their small size allows handling large numbers of fish in reduced space. Thus, zebrafish models enable high-throughput assays with significant statistical power, cost-efficient experiments and fewer ethical issues than murine models. The optical transparency of embryos allows direct visualization of development and makes the imaging and tracking of injected tumor cells possible without the need of invasive techniques . Its traZebrafish, or murine, xenograft tumor models are commonly used in cancer research and are very useful for testing new drugs. However, these models present limitations related to the lack of a fully functional immune system as required to favor the implantation and growth of tumor cells in other species. Zebrafish complete their basic development in the first 24 h post-fertilization (hpf) but they lack an adaptive immune system until the first 12 to 14 days of development, thus allowing this window for the implantation and progression of the cancer cells, without the need to use immunocompromised fish . DespiteNevertheless, xenografts are not applicable for every tumor type. Due to the phylogenetic distance between teleost fish and mammals, not all human organs are present in zebrafish. In order to overcome this hurdle, it has been suggested that analogous structures, such as gills could be used as a substitute for lungs . With reα/ARF are not expressed in zebrafish which rules out studies related to these pathways [Concerning genetics, the release of the sequence assembly of the zebrafish genome has revealed that around 70% of human genes are shared in zebrafish with more than 80% of them being human disease-related genes . Moreovepathways . Similarpathways ,113.Taking these pros and cons into consideration, below we describe relevant genetic and transplantation strategies that have been investigated using zebrafish as a model for GBM.G12V, HRASG12V and BRAFV600E) providing information about the role of selected genes in tumorigenesis [mpx:GFP) for neutrophils [mpeg1:EGFP) or Tg(mpeg1:mCherry) for macrophages [Genetic approaches for disease modeling in zebrafish comprise several strategies ranging from gene-targeted mutations to transient or stable over-/downregulation of genes of interest. Numerous cancer models have been established by transgenic expression of human and murine oncogenes in zebrafish  led to malignant tumors in the cranial cavity and parenchymal brain tumors respectively, highlighting that different tumor initiating cells may well determine the tumor type and demonstrating that tumorigenesis is driven by the activation of the canonical Ras and mTOR pathways [G12V, YAPS5A, KRASG12V, AKT, EGFRvIII and BRAFV600E oncogenes under the control of the zic4 enhancer, proliferating domain of developing nervous system. The somatic expression of activated RAS promoted brain tumors and/or heterotopia, suggesting their possible origin from a benign developmental lesion. Analyses of global RNA expression revealed that developed tumors resembled mesenchymal GBMs with a strong YAP component, suggesting YAP as a hallmark of malignant brain tumor and providing a useful model for preclinical drug screening for this GBM subtype [To model GBM-like tumors in zebrafish, several genetic approaches have been used . To illupromoter . In zebrpathways . Along t subtype . As soma subtype .Transient genetic modifications in zebrafish are commonly performed by the morpholino-based (MO) expression silencing, which enables the binding of the oligonucleotide to a desired target, knocking down the gene without sequence modification . MOs havDysregulation of cellular energetics and alteration of tumor cell metabolism, in particular glutamine metabolism, has been investigated using zebrafish models. For instance, glutamate dehydrogenase 2 (GLUD2) mRNA injection in zebrafish resulted in glial cell proliferation impairment while neuronal development was not affected, suggesting a novel potential drug target for GBM progression . AdditioOther mutagenesis approaches used to generate stable zebrafish cancer models include: Targeting Induced Local Lesions in Genomes (TILLING), based on the exposure to the mutagen ethyl-nitrosourea (ENU), Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), both based on nucleases and, most recently, CRISPR/Cas9 technology. These approaches have been used to demonstrate that NF1 loss increases penetrance of high-grade gliomas and malignant peripheral nerve sheath tumors in zebrafish with downregulation in telomerase and hyperactivation of ERK and mTOR pathways , as has 2+-dependent ATP release and its coupling with the microglia P2ry12 receptor, inducing their proliferation. This infiltration did not result in enhanced phagocytic activity of the microglia towards the preneoplastic cells even though that is the primary role of the cells in the central nervous system [A larval zebrafish model mimicking the earliest stages of brain tumor growth has been developed by overexpressing a cancer-promoting version of the human AKT1 oncogene in neural cells, under the neural-specific beta tubulin promoter, using the LexPR transcriptional activator system combined with macrophage and microglia reporters. In response to the activation of this oncogene, an increase in macrophage and microglia cell numbers was found in the brain, mediated by the SDF1b-CXCR4b signaling pathway which interacts with neoplastic cells by way of Cas system ,134. Furs system . Along ts system . It is ws system . Accordis system . These fAs a whole, genetic zebrafish models have been successfully used to study brain tumors from their initial stages of development, offering the possibility to manipulate selected pathways relevant in glioma and immune-related cells , to study their influence on the progression of the disease and also to look into the efficacy of their therapeutic intervention. Zebrafish xenografts commonly consist of the implantation of labeled cell lines of human or murine origin, or patient-derived cells into appropriate anatomical sites to generate in vivo heterotopic or orthotopic tumor models, which allow the tracking of a cancer cell’s survival, proliferation, metastasis, angiogenic potential, interaction with the microenvironment and response or resistance to new treatments ,139,140.prkdc−/−, il2rga−/−). Tumor cells robustly engrafted and proliferated showing similar growth kinetics and histopathology to those observed in a mouse model. Nevertheless, in this case, the requirement to pre-treat animals with clodronate liposomes in order to inhibit early macrophage ingestion might be too toxic, restricting the usefulness of this model [fli1:EGFP) embryos injected in the yolk sac with rat GBM-labeled cells (GV1A1) [vegfa and cyclin D1 expression were observed in 85% of the embryos with NO production, and the addition of a NO scavenger (CPTIO) reduced vegfa and cyclin D1 expression as well as the number of endothelial cells, suggesting that the reduction in NO levels by nitric oxide scavenging could be an efficient approach for the treatment of glioma. Xenograft models have provided information about GBM initiation and progression  and haveis model . The injis model . The imp (GV1A1) . Cells wfli1a:eGFP) casper embryos at 3 dpf and observed the attachment of cells to the brain vasculature within 24 hpi, as well as their expansion and migration along the vascular network by 7 dpf [irf8−/−), which does not contain microglia [irf8−/− decreased proliferation and survival, demonstrating the protumoral activity of microglia and how important it is to consider their differential response to distinct cell types when developing immunotherapies to treat gliomas. TGF-β was also shown to enhance tumor-induced angiogenesis via the JNK pathway and macrophage infiltration in a zebrafish glioma xenograft model [fli:GFP) embryos at 2dpf led to a significant increase in newly-formed blood vessels, as well as in macrophage accumulation in the brain region, tail and yolk sac. Additionally, the treatment of xenografted embryos with a JNK inhibitor resulted in a decrease in angiogenesis, which may well be a powerful model for anti-angiogenesis drug screenings. The same group established a tumor invasion model in 2dpf Tg(fli:EGFP) embryos by transplanting the U87 cell line and its derived cancer stem cells (CSCs) [The mechanisms of glioma-vessel interactions, angiogenesis and tumor invasion have also been studied in zebrafish models. Umans et al. intracranially injected an adult glioma cell line and a pediatric patient derived xenoline into Tg . CSCs enkdr:mCherry) embryos with labeled endothelial cells, harboring control scramble short haipin (shRNA), Rac shRNAs (shRacs) and Rac 1–3 cDNAs, to silence or overexpress Rac proteins respectively [RECQ1 in U87 cells transplanted into 2 dpf zebrafish brains resulted in decreased tumor growth. MOs were used to knock down lysine (K)-specific methyltransferase 2A (KMT2A), involved in glioma progression [fli1:EGFP) zebrafish brains increased both tumor growth and angiogenesis, even when embryos were treated with an immune suppressant (dexamethasone), revealing that KMT2A acts negatively on tumor growth in contrast with the previous conception of KMT2A as an oncogene in tumorigenesis [lama5), an important component of blood vessels, decreases GBM cell invasion and promotes the formation of blood vessel dependent microtumors. With 4D individual cell tracking technology and U251MG cells, xenotransplantation in the hindbrain ventricle of Tg(fli1:EGFP) 2 dpf embryos, proliferation, dispersion, microtumor formation and cell/blood vessel association were confirmed [lama5 knockdown by MOs resulted in significantly higher cell dispersion and mobility whereas microtumor formation was lower. Thus, lama5 increases GBM cell attachment to blood vessels by elevating VEGF activity, which in turn suppresses invasion but increases tumor formation.To examine the effects of Rac proteins on GBM progression in vivo, Lai et al. injected GFP-labeled U373- tumor sphere cells (GBM stem-like cells) in the yolk sac of 2 dpf Tg, the same NPs inhibited the growth of intersegmental vessels (ISVs) thanks to a controlled release of both drugs, in a more effective way than NPs loaded with just one of the treatments [Zebrafish xenotransplants have allowed GBM growth and response to treatment to be studied in real time, which, in turn, enables an evaluation of the proliferative, migratory, invasive and angiogenic status of the tumor. In these studies, TMZ was commonly used as the reference treatment and for positive control. For example, 2-methoxy-6-acetyl-7methyljuglone (MAM), a natural product that induced necroptosis in colon and lung cancer cells, injected into U251-xenograft zebrafish models showed similar anti-tumoral efficacy to TMZ at very low doses . A compaeatments . Zebrafieatments .Genetic and epigenetic therapies have been also tested using zebrafish GBM models. Protein arginine methyltransferase 5 (PRMT5) inhibitors, which regulate gene expression by methylation of histonic and non-histonic proteins, improved survival after microinjection of GBM human derived cells expressing GFP in the midbrain-hindbrain of 36hpf casper zebrafish . SchnekeZebrafish models have been applied to evaluate the outcomes of metabolic manipulation in cancer progression and GBM . ShtraizInhibition of angiogenesis is applied for the treatment of cancer to reduce the supply of oxygen and nutrients to the tumor, commonly in combination with other therapies . SeveralA major limitation in the application of new drugs in the fight against GBM is their ability to cross the Blood–brain–barrier (BBB). This physiological barrier limits the passage of molecules from the circulatory system to the central nervous system (CNS) and is the cause of failure for 98% of the drugs that target the CNSs tested in clinical trials .Previous studies on the development and maturation of the BBB in zebrafish have shown a sophisticated BBB, which is functionally and structurally similar to that of higher vertebrates. Although it is not exactly the same, these studies suggest the use of zebrafish as an experimental model organism for BBB-penetrating drug screenings ,198. ConDoxorubicin (DOX), a chemotherapeutic commonly used in cancer and unable to cross the BBB, is encapsulated in an apoferritin nanocage to target the transferrin receptor 1 (TfR1) overexpressed in brain endothelial cells and GBM cells. These NPs, labeled with the fluorescent dye Cy5.5, upon injection in the hearts of zebrafish are able to cross the BBB, so are an interesting strategy against GBM . The samIn addition to the studies detailed above, zebrafish have been widely used for toxicity, biocompatibility and/or biodistribution screening of drugs, benefiting from its ease of use and ethical and economic advantages versus other animal models. For example, cobalt (III) bound to nimesulide (Co-NMS), a COX-2 inhibitor, has been proven to increase the effects of radiotherapy in GBM and its neurotoxicity has been evaluated in zebrafish models from the changes in neurobehaviour capacity (swimming activity and movement) and morphological abnormalities in the CNS from hematoxylin/eosin staining of histological sections of the brain. Reduction in brain function and development has been observed at Co-NMS concentrations higher than 10 µM, while 5 µM concentration has been considered to be safe; there has also been a more intense radiotherapy effect on GBM in terms of a rise in the generation of ROSs and mitochondria damage in tumor cells . SeveralAs a whole, several studies using zebrafish have been performed for large drug screenings and also to understand the mechanism of action of particular treatments in zebrafish GBM. Furthermore, optimized zebrafish models, described in In recent years, seminal studies on glioblastoma (GBM) have unraveled the interactions between tumor cells and immune cells from the initial steps and along the progression of the pathology. In this regard, macrophages/microglia, representing up to 30-50% of cells in some tumors, present protumoral properties and functions which can, in theory, be reprogrammed with appropriate treatments, not yet available in the clinic, as suggested by some pre-clinical results described in In this context, genetic and xenotransplant zebrafish models have been successfully used to generate new knowledge about GBM pathology and some studies have been initiated for screening of new treatments. While there is still some room for improvement related to the modeling of the human immune system and optimization of orthotopic models of GBM in zebrafish, satisfactory results have already been established for detailed understanding of gene and metabolic alterations of the disease. Overall, we expect that zebrafish models will be further exploited by taking into account their biological, ethical and economic advantages for the screening and comprehensive evaluation of new drugs to ultimately improve the treatment of patients with glioblastoma.


Non-alcohol-related fatty liver disease (NAFLD) is common, affecting approximately 25% of the general population. The evidence base for the investigation and management of NAFLD is large and growing, but there is currently little practical guidance to support development of services and delivery of care. To address this, we have produced a series of evidence-based quality standard recommendations for the management of NAFLD, with the aim of driving improvement in patient care. A multidisciplinary group of experts from the British Association for the Study of the Liver (BASL) and British Society of Gastroenterology (BSG) NAFLD Special Interest Group (SIG) produced the recommendations covering: 1. Management of people with, or at risk of, NAFLD before the gastroenterology or liver clinic; 2. Assessment and investigations in secondary care, and 3. Management in secondary care. The quality of evidence for each recommendation was evaluated by the Grading of Recommendation Assessment, Development and Evaluation (GRADE) tool. An anonymous modified Delphi voting process was conducted individually by each member of the group to assess the level of agreement with each statement. Statements were included where agreement was ≥80%. From the final list of statements, a smaller number of auditable key performance indicators (KPIs) were selected to allow services to benchmark their practice. It is hoped that services will review their practice against our recommendations and KPIs and institute service development where needed to improve the care of patients with NAFLD. The disease ranges from steatosis to non-alcohol-related steatohepatitis  and can progress to cirrhosis and liver-related complications including hepatocellular carcinoma (HCC) and liver failure 2. Individuals with NAFLD have an increased risk of overall mortality compared with the general population, and common causes of death include cardiovascular disease, malignancy and liver-related complications 5. The majority of the estimated 14.1 million individuals with NAFLD in the UK remain undiagnosed and worryingly the prevalence of advanced fibrosis/cirrhosis is projected to double to 1 million individuals by 2030 6. Despite the high prevalence of NAFLD in the population, recognition and management of the condition is variable across the UK. One multicentre study from England found a large variability in the primary and secondary care management of NAFLD, with clear deficiencies identified in primary care investigations, fibrosis staging, provision of lifestyle treatments, and assessment and management of cardiovascular risk factors 7. There is therefore a clear need to improve the holistic management of patients with NAFLD in order to achieve better outcomes.Non-alcohol-related fatty liver disease (NAFLD) is common, affecting approximately 25% of the population in many developed countries The purpose of this work was to develop a series of quality standard recommendations from a multidisciplinary panel of experts for the management of patients with NAFLD to provide a standardised management approach, with the ultimate objective of reducing variability in care nationally. In addition, we have developed a series of auditable key performance indicators (KPIs) to measure practice against to help drive service improvement.A group of experts from the British Association for the Study of the Liver (BASL) and British Society of Gastroenterology (BSG) NAFLD Special Interest Group (SIG) developed the recommendations. SM and WA chaired the group. All members of the NAFLD SIG were invited to participate via email and those expressing an interest were included in the working group. Ultimately, the working group included a multidisciplinary team of 29 individuals from hepatology, diabetes, dietetics, hepatology specialist nursing, pathology, primary care, psychology, pharmacy and physiotherapy. The group also included a representative from the British Liver Trust (VH). The group was sub-divided into three working groups that led the writing of draft recommendations for one of three parts of the document: 1. Management of people with, or at risk of, NAFLD before the gastroenterology or liver clinic (lead LC); 2. Assessment and investigations in secondary care (lead MJA), and; 3. Management in secondary care (lead JFC).Each group produced a list of important topics in the NAFLD diagnosis and management pathway to address within the standards document. A literature search was conducted using PubMed Medline to identify relevant original research papers and existing guidelines to end of June 2021. The specific statements were then made by each group, informed by the quality of the evidence evaluated in line with the Grading of Recommendation Assessment, Development and Evaluation (GRADE) tool. SM and WA amalgamated the draft statements from the three working parties and removed any duplication. An anonymous modified Delphi voting process was conducted individually by each member of the working group using an online survey tool to assess the level of agreement with each statement on a five-point scale . Given the working group was multidisciplinary, members could abstain from questions that related to areas out with their usual clinical practice . After each round of voting, statements were redrafted where necessary through a combination of discussions via teleconference meetings and email. ‘Agreement’ was defined when statements received a score of ‘strongly agree’ or ‘agree’. Statements were included where agreement was ≥80%, after exclusion of any abstains. The result of this process produced a series of recommendations, with a corresponding level of expert agreement and grading of the relevant evidence.From this final list of statements, a smaller number of auditable KPIs were selected to allow services to benchmark their practice. The KPIs were chosen based on their potential to influence patient outcomes as well as being easily measurable.Following the Delphi voting process and the review of evidence, 34 quality standard recommendations  were mad9. Therefore, services should have an agreed local clinical pathway for the investigation of suspected liver disease that includes an assessment for liver fibrosis using available non-invasive liver fibrosis tests  or the metabolic syndrome  14. The ‘Scarred Liver Project’ 15, which offered community fibrosis testing to people with diabetes or obesity alongside those with hazardous alcohol consumption, identified 3688 patients at risk from a cohort of 25,018. Overall, 20% of at-risk individuals who attended a follow-up clinic had evidence of significant liver disease. Furthermore, a study including FIB-4 (discussed below) testing in annual diabetic reviews in primary care followed by transient elastography in those with an indeterminate or high FIB-4 found that 4.5% of the cohort had previously undiagnosed advanced liver disease, defined as imaging, endoscopic or biopsy evidence of cirrhosis, portal hypertension or HCC 16.Proactive assessment for the presence of liver fibrosis in patients at risk can permit earlier identification of significant liver disease 18. However, it is likely that a fibrosis risk-based approach in primary care may be more successful - and cost-effective - for the early identification of liver disease, than reliance on abnormal liver function or incidental finding of steatosis on imaging 19. However, it is also important to acknowledge that greater efforts to investigate and identify advanced liver disease in this group may result in a significant increase in primary and secondary care workload, and local service development considerations should be planned accordingly.Evidence to support a case finding strategy amongst people with significant risk factors is currently limited and remains an area of divergence between current European and North American clinical guidelines 18. In a study of 223 patients with biopsy-proven NAFLD, ALT more than two times the upper limit of normal (> 70 IU/L) had a sensitivity of just 50% and specificity of 61% for NASH, and sensitivity 40% and specificity 58% for advanced fibrosis 20. Moreover, the serum ALT level typically falls as liver fibrosis progresses and patients with cirrhosis frequently have a normal range ALT level 21. Therefore, do not rely on abnormal liver blood tests to prompt consideration of liver disease. However, persistently unexplained abnormal liver blood tests should always be investigated. , alkaline phosphatase (ALP), bilirubin and gamma glutamyl transferase (GGT), are frequently tested as part of routine clinical investigation in primary care. Whilst unexplained persistently abnormal liver blood tests should always be investigated, normal liver blood tests do not exclude NAFLD or significant fibrosis 22, with a focussed history eliciting risk factors for chronic liver disease including unhealthy alcohol consumption, the presence of metabolic risk factors and a detailed drug history. Automated systems, such as intelligent liver function tests (iLFT), can streamline the investigation of abnormal liver blood tests giving a full panel of results, suggested diagnosis and advice for further management 8. This approach increases the diagnosis of liver disease and is highly cost effective.Patients with abnormal liver blood tests should be evaluated in line with national recommendations 4. Patients with advanced fibrosis and cirrhosis  are at increased risk of complications of chronic liver disease, decompensation, liver transplantation and death in the short- to medium-term 25.An increasing body of evidence has demonstrated advancing liver fibrosis as the key predictor of liver-related events and mortality in patients with NAFLD 22. Accordingly, we suggest the finding of liver steatosis on ultrasound, or unexplained abnormal liver blood tests, should prompt risk assessment for liver fibrosis  or NFS <-1.455 (or < 0.12 if over 65 years) have a low probability of having advanced fibrosis and can be reassured 30. Of note, neither the FIB-4 nor the NFS have been validated in patients under 35 years of age 29. Evidence for the performance of other biomarkers such as Enhanced Liver Fibrosis test (ELF) or transient elastography (discussed below) in young patient cohorts is also lacking so non-invasive tests should be interpreted with caution in this age group.Indirect biomarkers of liver fibrosis such as FIB-4 and NAFLD Fibrosis score have been shown to have prognostic value for long term outcomes and are validated against liver biopsy with good negative predictive value to rule-out advanced liver fibrosis in NAFLD 31. The Camden and Islington NAFLD pathway stratified fibrosis in over 1450 patients with NAFLD over two years using FIB-4 in all patients and ELF test in FIB-4 indeterminate cases 9. Patients identified as high-risk for advanced fibrosis and cirrhosis were re-assessed in secondary care and resulted in a 5-fold increase in detection of advanced fibrosis, 3-fold increase in the detection of cirrhosis and 81% reduction in referrals of patients with mild disease in comparison to the absence of any defined care pathway.Exemplar pathways exist as recently reviewed 34.A number of cost-effectiveness studies have demonstrated the benefit of such risk stratification strategies. The use of serum markers and elastography, either alone or in combination has been shown to be clinically effective and cost saving compared to standard care Patients stratified as high risk for advanced fibrosis or cirrhosis whose liver disease is not outweighed by comorbidities or performance status should be referred to a hepatologist or gastroenterologist with an interest in liver disease for further evaluation of their condition.22. Although the optimum pathway has not yet been determined, liver stiffness measurement (LSM) by transient elastography > 8 kPa or an ELF > 9.5 has been used to prompt referral to secondary care liver services among existing pathways 32For patients stratified as indeterminate risk for advanced fibrosis or cirrhosis using simple non-invasive tests (NIT), the result is neither sensitive nor specific enough to confidently rule in or rule out advanced fibrosis or cirrhosis. A second tier liver fibrosis test such as a direct collagen biomarker  or elastography  should be offered 32.Ideally, second-stage non-invasive testing should be delivered in the community to reduce the unnecessary referral to secondary care for some individuals with a false positive simple NIT. However, where these services are not available in the community, patients should be referred to secondary services for further workup 4. NITs have high negative predictive values for advanced fibrosis so can reliably exclude the presence of cirrhosis. Moreover, long-term follow-up studies have shown that patients with low-risk NITs have a very low risk of liver-related events in the short- to medium-term, and their main morbidity is cardiovascular disease and non-hepatic malignancy 35. One recent long term follow up study of 1057 patients showed that individuals with NAFLD and a FIB-4 score <1.3 had a very low incidence rate for liver-related events of 2.6 per 1000 patient years 36. Therefore, for these patients the focus should be on lifestyle advice and cardiovascular risk reduction with the aim of improving their overall quality and length of life , illicit drug and smoking history documented  and undergo investigations for other causes of liver disease (i.e. completion of blood aetiology screen) if not already undertaken in primary care , anticonvulsants , analgesics (NSAIDs), glucocorticoids, anti-metabolites , oestrogen receptor modulators (tamoxifen) and antiretrovirals (efavirenz) 45. Initial assessment of patients with suspected NAFLD should therefore include a drug history, with consideration given to whether medication may be either the precipitant or a cofactor for steatosis. In addition, documenting a smoking history is important, especially given that cigarette smoking is associated with progressive fibrosis and cardiovascular disease 47Less commonly other drugs may be the precipitant for hepatic steatosis, with around 2% of cases of NAFLD attributable to prescribed medication 22. Genotype 3 strains of viral hepatitis C are associated with increased rates of steatosis 48, reinforcing the need for viral serology as part of a liver screen for all patients undergoing evaluation for suspected NAFLD.A comprehensive metabolic and serological screen should be undertaken  to consolidate the diagnosis of NAFLD and exclude co-existent liver disease 49. Therefore, a review of prescribed medications, use of over-the-counter medications and alternative/complementary medicines should be undertaken. As discussed above, commonly prescribed agents used to treat other conditions may contribute to hepatic fat accumulation  or accelerate progression (e.g. methotrexate). Whilst the published literature is at times conflicting, capturing use of methotrexate (including duration of exposure and cumulative dose) is particularly relevant as it is a potential cofactor promoting presence of a persistent transaminitis and/or an increased risk of advanced fibrosis/cirrhosis in patients who are overweight or diabetic 51. Consider recommending the discontinuation of relevant hepatotoxic medications after risk assessment, involving other relevant specialists as necessary. Moreover, it is important to conduct a medicines use review because discrepancies between patient-reported and medical record documented medications exist in more than 50% of patients with liver disease, particularly those taking more than five medications 52. Accordingly, we suggest practitioners should document a treatment history and medicines use review. The rationalisation of medicines that may accelerate disease progression should be considered. , further reduces the number of cases with an indeterminate score, allowing liver biopsy to be reserved for use in a minority of patients where it adds additional useful information 70. Such two-stage care pathways are currently considered to provide the most robust means for the risk-stratification of patients with NAFLD 71. Ongoing regular non-invasive fibrosis reassessment should be considered every 1-3 years to monitor response to treatment or for fibrosis progression 38.Patients with NAFLD should undergo sequential use of a simple non-invasive test (e.g. FIB-4) and specialist non-invasive tests  to assess the severity of fibrosis. , thereby necessitating a biopsy to understand their relative contributions to the patient’s condition 75.Liver biopsy remains the standard for diagnosing NASH and assessing disease activity  as there are no approved non-invasive radiological or serological markers specific for NASH. International drug authorities (FDA and EMA) therefore continue to recommend that phase 2/3 clinical trials utilise liver biopsy to confirm the diagnosis and assess the grade and stage of the disease, to determine trial entry, as well as act as a primary trial endpoint. There has been a recent debate about the role and reliability of histology in this setting In the event that there is discordance between non-invasive fibrosis markers, a biopsy may be required to stage fibrosis; and most importantly rule in or out cirrhosis.Patients with NAFLD should therefore be considered for a liver biopsy: A. if there is diagnostic uncertainty (other aetiologies/overlap conditions); B. to evaluate the severity of NASH and be considered for potential drug therapies ; C. to determine the stage of liver fibrosis where non-invasive tests are inconclusive to aid with future management   or steatosis, activity fibrosis (SAF) score with the choice of score being made by agreement with the clinicians 78. Accordingly, liver biopsies should be processed, stained and examined according to the RCPath guidelines and reported by pathologists who participate in the liver EQA scheme using a validated score such as the NASH CRN or SAF score.  or the expanded Baveno VI criteria (LSM <25kPa and platelet count >110 x109/L) have been validated in NAFLD 84, allowing endoscopy to be safely avoided in these selected patients. At present there is no prospective evidence to support screening for HCC in patients with NAFLD without cirrhosis. Accordingly, we suggest patients with NAFLD cirrhosis should be offered surveillance for complications of cirrhosis, including HCC and varices, in accordance with national/international recommendations. The Baveno VI exclusion criteria should be considered as a non-invasive tool to rule out the presence of varices requiring treatment.  is the leading cause of death among patients with NAFLD, accounting for over a third of the deaths 5. The risk of CVD mortality increases with disease severity, with higher rates in those with biopsy-confirmed NASH (2-fold) and in particular advanced fibrosis 87. Even though debate remains over the true causal relationship between NAFLD and CVD 88, the overall consensus is that traditional cardiovascular risk factors should be actively assessed and modified (where possible) in order to improve clinical outcomes in patients with NAFLD. Modifiable risk factors of CVD include smoking, hypertension, high non-high-density lipoprotein (HDL) cholesterol, lack of physical activity, unhealthy diet, alcohol intake above recommended levels and overweight/obesity. In addition, there should be an increased awareness of non-modifiable risk factors including older age, male gender, family history of CVD, and ethnic background (esp. South Asian origin) when evaluating CVD risk in patients with NAFLD 89.Patients with NAFLD have increased cardiovascular-related morbidity and mortality, largely as a result of the association between NAFLD and the metabolic syndrome 89 currently recommend the QRISK-3 assessment tool (which includes ethnicity) in all individuals over 40 years of age who have no prior history of CVD. Although these guidelines are for asymptomatic individuals and are yet to be validated in patients with NAFLD, there is no reason to suggest that QRISK-3 would not be applicable in NAFLD clinics to guide primary prevention with pharmacological therapy  90. Therefore, people with NAFLD should undergo systematic assessment of cardiovascular risk factors including use of an objective risk score   have been suggested over the years to estimate risk of CVD at 10 years in the general population. UK guidelines 92. We therefore suggest that patients with NAFLD should be screened annually for Type 2 Diabetes (using HbA1c), hypertension and dyslipidaemia  93. NICE guidelines recommend annual screening for T2DM amongst individuals with risk factors and evidence of an elevated plasma fasting glucose and pre-diabetes (defined as an HbA1c 42-47 mmol/mol (6.0-6.4%))94. For those with an HbA1c of less than 42 mmol/mol they recommend a reassessment in 3 years 94. Given NAFLD is recognised as a high-risk group for T2DM 34 and for simplicity we advocate annual screening for diabetes in patients with NAFLD. A HbA1c of 48 mmol/mol (6.5%) or above is diagnostic for T2DM 95. The utility and convenience of blood testing for HbA1c in the out-patient setting, favours its use over that of fasting glucose sampling and/or 75g oral glucose tolerance testNAFLD has a strong relationship with multi-organ insulin resistance, most notably the liver, muscle and adipose tissue. NAFLD is associated with a 2-5 fold increased risk of developing T2DM after adjustment of several metabolic and lifestyle confounders 96. In addition, many prospective studies have shown that NAFLD is an independent risk factor for systemic hypertension (3-fold increase versus non-NAFLD) 97, which itself when left uncontrolled (clinic blood pressure >130/85) is a major risk of all-cause and CVD-related mortality 98. Not only are these metabolic conditions treatable , but they can be used as clinical markers to predict those patients with NAFLD who are at risk of underlying NASH and progressive fibrosis 99.Patients with NAFLD are at risk of proatherogenic dyslipidaemia characterized by high triglycerides, increased very-low- density lipoprotein (LDL), and a higher concentration of small dense LDL coupled with low high-density lipoprotein (HDL) concentrations 5. Tobacco smoking markedly increases risk of cardiovascular, neoplastic and respiratory diseases, leading to increased all-cause morbidity and mortality. Smoking cessation dramatically reduces age-specific mortality rates 100. People with NAFLD should be asked about their current and past cigarette smoking history. People who currently smoke should be advised to stop and offered assistance in stopping and referred to smoking cessation services 101.People with NAFLD should be asked about smoking and, if they smoke, should be advised to stop and offered referral to smoking cessation services.  and a quantitative alcohol history is essential for diagnosis 106. As cofactors, both alcohol use and obesity have been shown to correlate with progression of portal hypertension in chronic liver disease 108.The transition from compensated to decompensated disease is associated with greatly increased morbidity and mortality. In people with alcohol-related liver disease, continued drinking is a stronger risk factor for decompensation than any histological or laboratory parameters 34. However, alcohol can be a source of additional dietary calories 109 and minimising use may avoid further weight gain with worsening of metabolic risk factors. Accordingly, abstinence from alcohol should be strongly recommended to patients with NAFLD and cirrhosis. Patients with pre-cirrhotic NAFLD should be advised that alcohol consumption may accelerate disease progression and so should minimise or abstain from alcohol to reduce the risk of disease progression.  on morbidity and mortality, there is robust histological evidence to demonstrate improvements in NAFLD liver histology 116. Bariatric surgery in patients with NASH can result in NASH resolution; a prospective study of 109 individuals with NASH found 85%  had resolution on biopsy a year post-bariatric surgery 117. A more recent meta-analysis found obesity surgery improves steatosis and steatohepatitis in 88%  and 59%  of patients respectively, and fibrosis in 30% (95% CI 21-41) 118. Furthermore, bariatric surgery also improves overall mortality from cardiovascular and malignant causes.Referral for consideration of bariatric surgery should be considered in NAFLD patients with obesity and who meet the eligibility criteria for bariatric surgery according to national recommendations , advanced fibrosis (F3) or cirrhosis (F4) are at risk of progression to end stage liver disease and potential complications in the medium to long-term 124. Factors that may influence the decision to refer for assessment include the presence of life-limiting comorbidities or recent (within 5 years) extrahepatic malignancy (except skin) that would be contraindications to transplantation.Patients with decompensated liver disease caused by NAFLD should be considered for transplant assessment  should have cardiovascular risk assessment using QRISK3 assessment tool, and those with 10% or greater 10-year risk of developing CVD should be offered HMG-CoA reductase inhibitor (“statin”) therapy for primary prevention 8991.Despite the risk of progressive liver disease, the leading cause of death in patients with NAFLD is CVD 130, their use in NAFLD is sometimes limited by concerns about hepatotoxicity. A pairwise meta-analysis of over 120,000 people in whom the presence of NAFLD was not recorded reported a small increase in liver dysfunction (OR 1.33 (1.12- 1.58)) overall, but these and other adverse effects did not outweigh the reduction in risk of major cardiovascular events 131. Conversely, there is evidence of benefit in the context of liver disease: in patients undergoing biopsy for suspected NASH, statin use conferred dose-dependent protection against liver-related histological endpoints, including steatohepatitis and fibrosis 132. A cross-sectional study of individuals with biopsy-proven NAFLD and T2DM demonstrated that statins were negatively associated with steatohepatitis and significant fibrosis in multivariate analyses 133. A recent meta-analysis of eight studies including patients with mixed aetiologies of cirrhosis (n=3195) concluded that statin use was associated with an improvement in portal pressure gradients and a reduced risk of variceal haemorrhage 134.While statins confer survival benefit in both primary and secondary prophylaxis of cardiovascular events Therefore, we recommend patients who are at increased cardiovascular risk (T2DM and/or QRISK-3 >10%) should be offered HMG-CoA reductase inhibitor (“statin”) treatment in accordance with NICE guidelines  and sodium-glucose co-transporter 2 inhibitors (SGLT2i) 139. NAFLD is a relatively recently described entity with substantial unmet need in terms of understanding of its natural history, diagnostic tests, and treatment to prevent disease progression. These needs can only be addressed through the engagement of people with NAFLD with research. Where research studies are available, these should be offered to appropriate patients to consider participation.Patients with NAFLD should be considered for research studies and offered the opportunity to participate in clinical trials where available   143 and the NAFLD patient guidelines from EASL 144Patients should be provided with written information about NAFLD and weight management in a format appropriate to their needs and signposted to other credible sources of information such as the NHS and the British Liver Trust (Recommendation 34, The evidence base for investigation and management of NAFLD is large and growing, but there is currently little practical guidance to support development of services and delivery of care. To address this, we have produced a series of evidence-based quality standard recommendations for the management of NAFLD, with the aim of driving improvement of the care of patients with this common condition. Currently there is no high-quality evidence for liver specific management in NAFLD, so the recommendations are likely to evolve as evidence accumulates. It is hoped that services will review their practice against the KPIs and institute service development where needed. The NAFLD SIG will aim to conduct a national audit of the management of NAFLD using the KPIs as a benchmark to promote service development.


While combined antiretroviral therapy (cART) allows near-normal life expectancy for people living with human immunodeficiency virus (HIV), it is unable to cure the infection and so life long treatment is required.The main barrier to curing HIV is the latent reservoir of cells, which is stable and resistant to cART.Current approaches under investigation for clearing this reservoir propose a ‘Shock and Kill’ mechanism, in which active replication is induced in latent cells by latency reversal agents, theoretically allowing killing of the newly active cells.T central memory cell reservoir, are unable to target other latent reservoirs and may be causing neurological damage to participants.However, previous studies have failed to achieve depletion of the in vitro and ex vivo studies, and may represent the basis of a future functional cure for HIV.Future approaches to clearing the latent reservoir may bypass latency reversal through the use of drugs that selectively induce apoptosis in infected cells. Several classes of these pro-apoptotic drugs have shown promise in  Furthermore, escape mutations of the virus are common and reduce efficacy,5 necessitating expensive combination therapy with multiple drugs. Viral rebound occurs due to the presence of latent viral reservoirs, rather than continuing low-level replication during cART, as shown by clonal evolutionary studies.6 On therapy cessation these latently infected cells clonally expand, seeding a population of infected cells containing intact provirus.7 Whilst cART has been successful in extending life expectancy of people living with HIV in high-income countries, this remains impaired compared to healthy adults.8 As cART is limited by mutations, is an expensive lifelong treatment and may not be available to those in countries with poorly developed healthcare systems, development of a cure for HIV is desirable. Cure research would be of considerable impact in South Africa, which has the highest number of people living with HIV in the world, at 7.7 m as of 2018, a prevalence of 20.4% in adults (UNAIDS 2019)9. In that year 77 000 deaths were recorded due to AIDS-related illnesses (UNAIDS 2019), although this number is coming down and life expectancy is rising as the cART programme continues to grow.The global HIV/AIDS pandemic has caused over 25 million deaths since its origins in the 20th century and remains without a cure. Combined antiretroviral therapy (cART) is the current treatment of choice for human immunodeficiency virus (HIV) infection+ central memory T cells (TCM) containing replication-competent, integrated provirus.10 These were found to be present in patients on cART, with no change in reservoir size with continuous therapy11 as the quiescent cells do not contain actively replicating virus, and so are unaffected by cART. The TCM- reservoir is present even if cART is initiated within the first week post exposure,12 so is established early in infection. It is also extremely stable, as the latent cells have a half-life of around 44 months,13 so is unlikely to be cleared in a patient’s lifetime. The TCM- reservoir contains the majority of latent cells, but other reservoirs are also present and form barriers to cure research. Several myeloid cell lines have been shown to harbour latent HIV in chronic infection and contribute to viral rebound on cART cessation.15 There is evidence for the presence of latent reservoirs in tissue macrophages16 and microglial cells and astrocytes in the central nervous system (CNS).18 There is no in vivo evidence of latently infected tissue dendritic cells but infection has been shown in vitro,19 and it has been proposed that this may be transferred to CD4+ cells via a virological synapse.20 Follicular dendritic cells have been shown to maintain a stable pool of virus on their surface without being infected, providing a latent reservoir within secondary lymphoid tissue.21 Further reservoirs have been suggested, such as within fibrocytes,20 but it is now apparent that the TCM reservoir is not the only source of latently infected cells. Potential cures must, therefore, target all identified reservoirs to be successful.Reservoirs of latently infected cells are the main barrier to cART curing HIV. The first latently infected cells to be identified were CD430 and may have unresearched deleterious side effects.33 An emerging class of drugs targeting the apoptosis pathways in latently infected cells may allow reservoir depletion without latency reversal, and therefore merit further research as a potential cure for HIV.Several approaches to clearing the latent reservoirs have been investigated, with the ‘Shock and Kill’ combination receiving particular interest. However, recent findings seem to suggest this is unlikely to ever provide a cure for HIV+ T cells, cART or other agent, resulting in clearance of the latent cells and curing the infection. This mechanism is outlined in in vitro model of latently infected CD4+ TCM34 to identify compounds that selectively induce viral replication in latent cells. These include disulfiram22 and histone deacetylase inhibitors (HDACIs) such as vorinostat and panobionstat. Early in vivo studies were promising as latency reversal was demonstrated with an increase in viral gene expression in the resting cells observed with disulfiram,24 vorinostat25 and panbionstat26 administration. However, the key caveat is that none of these studies decreased reservoir size in vivo as the number of latent cells did not decrease following latency reversal. Whilst replication was induced in a proportion of the latent cells, this did not lead to immune or viral-mediated killing of reactivated cells.A potential approach to clearing HIV latent reservoirs is ‘Shock and Kill’, in which the reservoir would be ‘shocked’ with latency-reversing agents (LRAs) to induce replication of the provirus. ‘Killing’ of the activated cells would then be achieved through viral cytopathic effects, CD827 showed that the viral cytopathic effect alone is insufficient to kill cells, and the vigorous CTL response is lost in chronic HIV infection. However, CTL stimulation increased latent cell clearance, leading to the suggestion that some form of immune stimulation may be required alongside latency reversal to achieve a reduction in reservoir size. The CTL response to reactivated cells may be insufficient to reduce reservoir size on latency reversal, due to 98% of latent T cells carrying escape mutations to CTL killing28 and T cell exhaustion in chronic infection29 reducing the efficacy of the CTL response. Furthermore, LRAs may not affect non-TCM- reservoirs30 as macrophages are resistant to the cytopathic effects of HIV, and dendritic cells do not integrate viral DNA, meaning that LRAs would be unable to cause viral replication and subsequent targeting by the immune system. It has also been proposed that continued cART may not completely inhibit infection of further cells by virions produced from reactivated, formerly latent cells.30 Thus, LRA administration may lead to infection of previously uninfected cells. It is therefore clear that LRA administration alone does not reduce reservoir size, due to a lack of viral cytopathic effects and lysis, insufficient CTL response and the potential failure of cART to suppress replication. As such, stages 3 and 4 from ex vivo35 or other agents, such as broadly neutralising antibodies (bNAbs), which appear to mediate antibody-dependent cellular cytotoxicity of activated latently infected cells in mice36 but human studies are required to assess their efficacy. Killing agents that selectively induce apoptosis of HIV-infected cells have also shown promise in vitro (see Pro-apoptotic drugs [PADs]).Several reasons have been proposed for the failure of LRAs alone to reduce reservoir size. The initial principle of ‘Shock and Kill’ was that viral cytopathic effects and lysis, due to HIV replication, aided by cytotoxic T lymphocyte (CTL)-mediated killing of actively infected cells, would be sufficient to kill reactivated, formerly latent, cells. Shan et al.37 However, it seems likely that even with killing agents LRAs cannot clear enough latent cells to achieve a functional cure and may cause complications due to non-T cell HIV reservoirs. Future studies in humans to test the efficacy of killing agents administrated alongside LRAs could be carried out by administering a previously tested LRA, such as disulfiram, alongside a killing agent such as a bNAb to participants living with HIV and measuring the change in reservoir size using a technique such as TILDA.38 However, the combination of an LRA with, for example, a bNAb, is unlikely to clear the myeloid reservoir as dendritic cells are likely to be unaffected by LRAs.30 Therefore, even if functional elimination of the TCM reservoir is achieved, viral rebound on cART cessation may still occur due to the presence of the myeloid reservoir, which would likely not be cleared by current ‘Shock and Kill’ approaches under investigation.Whilst it has been repeatedly shown that LRAs alone are unable to reduce the size of the latent HIV reservoir, administration of a killing agent alongside an LRA has been proposed as a method of clearing the reactivated cells.39 suggesting significant damage to the CNS even when viraemia elsewhere is suppressed below detectable levels. The cause of this appears to be relatively poor blood-brain barrier (BBB) penetrance by antiretrovirals,31 allowing continued HIV replication alongside the presence of latently infected microglial cells, perivascular macrophages and astrocytes.20 Recent research suggests that impaired neurogenesis may underpin HAND persistence even with cART.32 A study on the CNS penetrance of LRAs33 showed that disulfiram and vorinostat are able to cross the BBB relatively easily. Whilst this could enable targeting of the CNS latent reservoir, it may mean that current in vivo studies of LRAs are damaging to the patients involved, as there is no reduction in latent reservoir size, whilst LRAs are able to penetrate the CNS and increase viral replication with no inhibition from antiretroviral drugs. This may potentially lead to an increase in viral protein, inflammatory cytokine (CK) and neurotoxin release from infected macrophages and microglia, which could increase the probability of HAND development  as shown in Human immunodeficiency virus-associated neurological disorders (HANDs) are well-observed in patients on cART and may lead to widespread neurological impairment,40 In latently infected cells the increase in longevity observed is partially the result of avoidance of apoptosis.37 A number of specific effects on apoptosis pathways have been suggested as the cause of this. The intrinsic apoptosis pathway is a response to cellular damage and involves pro-apoptotic factors causing mitochondrial outer membrane permeabilisation (MOMP), triggering the release of soluble proteins from the mitochondrial intermembrane space. These include the cytochrome c and second mitochondria-derived activator of caspase . Cytochrome c binds cytosolic proteins to form the apoptosome, which activates the ‘initiator’ enzyme caspase-9. This, in turn, cleaves the ‘executioner’ enzymes caspase-3 and caspase-7 which trigger the cleavage cascade of other caspase enzymes resulting in cell death. The second mitochondria-derived activator of caspase activates this process through disinhibition of caspases 3, 7 and 9 via binding and inactivating the X-linked inhibitor of apoptosis (XIAP). The X-linked inhibitor of apoptosis binds these caspases in the absence of SMAC, inhibiting their activity and directing their degradation by the proteasome. The second mitochondria-derived activator of caspase antagonises this activity, allowing the progression of the apoptotic process.41 Several other IAPs are also inhibited by SMAC, including the baculoviral IAP repeat-containing protein 2 .41The human immunodeficiency virus has complex interactions with apoptosis pathways in infected cells.42 The X-linked inhibitor of apoptosis inhibits caspase activity, as detailed earlier, whilst Bcl2 inhibits MOMP,43 thus preventing the release of cytochrome c and SMAC. Tat also inhibits the pro-apoptotic proteins p53 and the Bcl2-associated antagonist of cell death (BAD).40 Nef causes p21-activated kinase activation, leading to phosphorylation of BAD, thus inhibiting its pro-apoptotic activity (binding to and inhibition of Bcl2),44 and also inhibits caspase-8 and caspase-10.40 Conversely, in the CNS, it appears that Tat and Nef may induce autophagy and apoptosis in HAND pathogenesis.46 Expression and activation of anti-apoptotic proteins are therefore increased in latently infected cells, making this an attractive therapeutic target.Viral proteins, in particular Tat and Nef, cause inhibition of this intrinsic apoptosis pathway in latently infected cells. Tat upregulates host production of anti-apoptotic proteins including XIAP and Bcl2.48 as tumour cells often over-express or activate IAPs allowing the evasion of apoptosis, a key hallmark of cancer. Some of these have been shown to induce apoptosis of latent HIV infected cells in vitro, leading to speculation that PADs may contribute to a future cure.Several classes of pro-apoptotic compounds have been developed recently for cancer treatment,48 They cause caspase activation via IAP binding49 and appear to reduce IAP activity through both degradation and inhibition of the proteins,50 and therefore induce apoptosis. Second mitochondrias have recently been developed for cancer treatment, inducing apoptosis48 of tumour cells which over-express or activate IAPs. The SMs birinapant, embelin and GDC-0152 were investigated for their efficacy to induce apoptosis in latently infected TCM by Campbell et al.51 All were found to cause rapid degradation of XIAP and BIRC2, which were upregulated in the infected cells, and importantly showed selective killing of HIV-infected TCM compared to uninfected TCM, with the doses required for 90% clearance of HIV-infected cells causing a small increase of TCM cell death, of 3.5% – 4.6% above basal levels. This occurred in the absence of increased virus production, despite findings in models of latency by Pache et al.52 suggesting that SMs may act as LRAs due to the reduction in BIRC2 leading to increased NFκβ signalling, resulting in an increased viral transcription. However, in latent HIV-TCM from patients undergoing cART, the Pache study showed that SMs only caused latency reversal when used in combination with an LRA such as vorinostat. Therefore, it appears that the major effect of SMs on latently infected TCM- -is to induce apoptosis rather than reactivate transcription.Second mitochondria-derived activator of caspase mimetics (SMs) are a class of drugs which bind IAPs in a similar fashion to the endogenous molecule.CM. Inhibitors of later stages of autophagy did not reduce apoptosis, suggesting that the process of autophagy itself is not required for this mechanism of cell death. Induction of autophagy is sufficient for apoptosis, as autophagy proteins allow DISC formation. IAPs are degraded by SMs in uninfected TCM but this does not lead to the same degree of cell death due to reduced autophagy induction and therefore decreased probability of DISC formation. HIV-TCM are more prone to autophagy induction than uninfected cells as a result of the effects of viral proteins. Gag and Nef interact with autophagy factors to increase autophagy induction, which in turn augments HIV yields in cells containing the actively replicating virus.53 Nef and Vif inhibit later stages of autophagy to prevent HIV degradation.54 Therefore, HIV-TCM are more prone to autophagy induction than uninfected cells, providing a potential explanation for the selective apoptosis observed in latently infected cells on SM administration. Over-expression or activation of IAPs in infected cells may also contribute to the selectivity of SMs.Furthermore, Campbell et al. demonstrated the mechanism of apoptosis on SM administration is dependent on the induction (but not completion) of autophagy. Wortmannin (an inhibitor of early stages of autophagy) led to a reduction in apoptosis despite the degradation of IAPs by SMs. Further findings suggest that, following IAP degradation, autophagy proteins form a scaffold for the assembly of a ripoptosome-like death-inducing signaling complex (DISC), which can then initiate apoptosis via caspase 8 induction. This is an additional mechanism to the simple removal of IAP inhibition due to their degradation and may explain the selectivity of SMs for inducing death of HIV-Tex vivo evidence of the selective killing of latently infected TCM by SMs, along with a convincing mechanism of how uninfected cells are spared. Clinical trials in humans may now be justified, but should be performed with caution as the side effects of these drugs in people living with HIV are unknown. A number of SMs have been used in phase I trials for various cancers, with a generally tolerable safety profile, although the occasional occurrence of cytokine release syndrome in patients is of some concern.55In vitro and ex vivo findings suggest SMs may be able to deplete the TCM reservoir in vivo, and, whilst if 90% of latently infected cells are killed (as in the Campbell study) the patient will not be cured of HIV, this eclipses reservoir clearance demonstrated by current ‘Shock and Kill’ approaches. However, it is unclear if these drugs would induce apoptosis in non-T cell reservoirs to the same degree, which may prevent the development of a functional cure. Future in vitro and ex vivo studies of PADs using latently infected myeloid cells from people living with HIV would be advisable to further test their suitability.These findings present In vitro and ex vivo studies of other PADs have also shown promise. Bcl-2 antagonists such as venetoclax caused disinhibition of MOMP and led to the depletion of latent HIV-TCM from patients on cART in an ex vivo study.56 P13K/Akt inhibitors increase the expression of pro-apoptotic genes downregulated by HIV and have shown promise in predisposing latently infected macrophages and microglia to cell death.58 RIG-1 inducers activate an innate immune response to viral RNA, triggering apoptosis. An ex vivo study demonstrated selective depletion of latent HIV-TCM using the RIG-1 inducer acitretin.59 These classes of PADs also merit further investigation and could be used in combination with SMs in a future approach to clearing the latent reservoir to induce apoptosis in a greater portion of latently infected cells.in vitro and ex vivo success in selectively causing apoptosis in latently infected cells, with SMs appearing effective against the TCM reservoir and P13K/Akt inhibitors against myeloid reservoirs. Several of these drugs have been used in clinical trials for other conditions, and further investigation into their potential for people living with HIV is merited.Recent ‘Shock and Kill’ research has failed to demonstrate significant depletion of the viral reservoir, and it remains unclear if the use of LRAs may cause HANDs. PADs may represent a promising future approach to curing HIV given their


Tbx20 is a member of the Tbx1 subfamily of T-box-containing genes and is known to play a variety of fundamental roles in cardiovascular development and homeostasis as well as cardiac remodeling in response to pathophysiological stresses. Mutations in TBX20 are widely associated with the complex spectrum of congenital heart defects (CHDs) in humans, which includes defects in chamber septation, chamber growth, and valvulogenesis. In addition, genetic variants of TBX20 have been found to be associated with dilated cardiomyopathy and heart arrhythmia. This broad spectrum of cardiac morphogenetic and functional defects is likely due to its broad expression pattern in multiple cardiogenic cell lineages and its critical regulation of transcriptional networks during cardiac development. In this review, we summarize recent findings in our general understanding of the role of Tbx20 in regulating several important aspects of cardiac development and homeostasis and heart function. Brachyury  is considered the most ancient gene of the family and is critical to the formation and development of the posterior mesoderm , plays an important role in early embryonic development, including mesodermal differentiation and migration as well as endoderm specification during gastrulation  and TBX2 subfamily  exhibit a comprehensive spatiotemporal expression pattern involved in all cardiogenic lineage cells, which are critical to cardiac development  family genes encode various transcription factors that are essential for embryonic development and organogenesis in the evolution of all metazoan, ranging from hydra to humans , and second heart field (SHF)  and neuromancer2 , is expressed in early cardioblasts of the dorsal vessel  analyses demonstrate that Tbx20 directly binds the conserved T-half sites within the Isl1 promoter sequence, suggesting that Tbx20 is a negative regulator of Isl1 expression , a member of the epidermal growth factor family, is known for its critical role in ventricular wall trabeculation  was also found to be a transcriptional repressor of Tbx20 expression. Decreased expression levels of Tfap2c can upregulate Tbx20 expression . Tbx20 is an important player in the modulation of extracellular matrix expression along with the promotion of cell proliferation in mesenchymal valve precursor structures derived from endocardial cushions  within TBX20 are linked to prolonged QRS duration with strong linkage disequilibrium (LD) , a critical channel responsible for ventricular repolarizing currents. The human TBX20 p.R311C mutation can lead to the loss of TBX20 transcriptional activity, which subsequently causes a lower expression level of hERG and inward rectifier current, leading to prolonged action potentials  in a spatiotemporally precise manner. The CCS is a complex set of specialized structures and cell types in the heart, which include the sinoatrial node (SAN), atrioventricular node (AVN), fast-conducting atrioventricular bundle (AVB), left and right bundle branches, and Purkinje fiber (PF) network. In addition, working cardiomyocytes also play a pivotal role in propagating electrical impulses throughout the myocardium. Congenital defects of the CCS and dysregulation of CCS homeostasis can lead to CCS dysfunction, causing life-threatening arrhythmias and increasing the risk of death in both children and adults (Wolf and Berul, In summary, this review summarized the recent findings related to our general understanding of the role of Tbx20 in regulating several important aspects of cardiac development, homeostasis, and heart function. These advances provide a basis for the early genetic diagnosis of associated CHDs, cardiomyopathies, and heart functional defects. However, the broad associations of Tbx20 with multiple biological events in multiple cardiogenic cell lineages during cardiac development make it difficult to map out all Tbx20-mediated upstream and downstream genetic networks intercellularly and/or intracellularly, especially the correlation of its genetic mutations found in complex CHD and cardiomyopathy patients with unique disease outcomes. Future analyses using hESC- and hiPSC-based analyses may help to confirm the findings from various animal models, thus addressing the key issues in disease progression in humans.YC drafted the manuscript. DX and LZ helped with early revision. C-LC, B-YL, and YL finalized the manuscript. All authors contributed to the article and approved the submitted version.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.


Allosteric modulators are ligands that bind to a site on the receptor that is spatially separated from the orthosteric binding site for the endogenous neurotransmitter. Allosteric modulators modulate the binding affinity, potency, and efficacy of orthosteric ligands. Muscarinic acetylcholine receptors are prototypical allosterically-modulated G-protein-coupled receptors. They are a potential therapeutic target for the treatment of psychiatric, neurologic, and internal diseases like schizophrenia, Alzheimer’s disease, Huntington disease, type 2 diabetes, or chronic pulmonary obstruction. Here, we reviewed the progress made during the last decade in our understanding of their mechanisms of binding, allosteric modulation, and in vivo actions in order to understand the translational impact of studying this important class of pharmacological agents. We overviewed newly developed allosteric modulators of muscarinic receptors as well as new spin-off ideas like bitopic ligands combining allosteric and orthosteric moieties and photo-switchable ligands based on bitopic agents. Detailed analysis of methoctramine binding has revealed two modes of interaction with the receptor. Transient binding to the common allosteric site center 1 (namely E175 in the o2 loop) is followed by stable binding that occurs concurrently to both the allosteric and orthosteric sites  has has5 mus3 receptor [3 PAM of acetylcholine VU0119498 (3 receptors in their β cells.In type 2 diabetes, pancreatic β cells are unable to release sufficient amounts of insulin to maintain physiological blood glucose levels. Parasympathetic activation of pancreatic β cells by acetylcholine stimulates the release of insulin . Effectsreceptor . In accoU0119498  has beenRecent findings of the diversity of actions of allosteric modulators and bitopic ligands of muscarinic receptors have greatly influenced the development of new therapies for a variety of disorders. Recent discoveries of selective positive allosteric modulators of acetylcholine with therapeutic potential in the treatment of psychiatric and neurologic disorders like Alzheimer’s or schizophrenia, as well as internal disorders like type 2 diabetes, are encouraging. Research on several muscarinic allosteric modulators has surpassed the stage of optimization of selectivity, efficacy, and bioavailability and advanced to a translational stage of medicinal research. In the field of basic research, further progress is mainly achieved by the introduction of novel concepts and techniques. Novel photo-switchable ligands may greatly increase the accuracy of timely delivery as well as allow varied permutations of experimental design. Thus, focus on the design of novel photo-switchable ligands is currently gaining momentum.


Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation. Regulated cell death (RCD), a form of cellular demise that is governed by a genetically encoded molecular machinery,Cellular stressors that are associated with ICD encompass (but are not limited to): (1) obligate intracellular pathogens including multiple bacterial and viral speciesThe aforementioned ICD inducers have been instrumental not only for identifying the molecular machinery that underlies the immunogenicity of some variants of RCD,Definition of immunogenic cell death), the presence of such signals is not necessarily predictive of the ability of dying cells to drive adaptive immunity in vivo.The morphological features displayed by dying cells and the molecular mechanisms that are mechanistically responsible for the cellular demise do not necessarily correlate with the immunogenicity of RCD.The Nomenclature Committee on Cell Death has recently defined ICD as ‘a form of RCD that is sufficient to activate an adaptive immune response in immunocompetent syngeneic hosts’,Sources of ICD antigenicity). Conversely, infected cells, as well as malignant cells, display sufficient antigenicity to drive immune responses, as they express a panel of antigenic epitopes for which naïve T cell clones are generally available. These antigenic determinants include neoepitopes that are highly immunogenic as they are not covered by central tolerance as well as (non-mutated) epitopes that may be immunogenic due to gaps in central tolerance and/or incomplete peripheral tolerance.Antigenicity is conferred by the expression and presentation of antigens that fail to induce clonal deletion in the context of central tolerance in a specific host, implying that the host contains naïve T cell clones that can recognize such antigens.Microenvironmental conditions also have a dramatic influence on the propensity of infected or neoplastic cells undergoing a potentially immunogenic variant of RCD to initiate adaptive immunity and/or be susceptible to CTL-dependent lysis, thus impacting both the priming and the effector phase of the immunological response. As an example, mouse cancer cells irradiated in vitro can be successfully employed to immunize immunocompetent syngeneic mice against a subsequent challenge with living cells of the same type, demonstrating the elicitation of immunological memory.Taken together, these observations suggest that while the adjuvanticity of RCD depends on dying cells and the capability of the initiating stressor to elicit danger signaling, its immunogenicity ultimately depends on dying cells and the host, which also determines the propensity of dying cells to drive adaptive immunity at the microenvironmental level .Infection by pathogenic microbes is an obvious source of antigenic determinants, as microbial proteins are not covered by central tolerance and hence their epitopes are highly antigenic.There are at least two exceptions to this principle, which may underlie the ability of healthy cells to undergo ICD. First, the genome of normal cells contains a significant number of endogenous retroviruses, which are generally latent  in physiological conditions.The majority of human tumors are not driven by active viral infections. Nonetheless, like pathogen-infected dying cells, malignant cells can display a high antigenicity, largely reflecting the elevated mutational rate that frequently accompanies malignant transformation and disease progression in the context of immunoevasion.Detection of ICD in cancer). Second, the antigenicity of malignant cells is directly related to antigen presentation, implying that genetic and epigenetic defects that compromise it can be beneficial for cancer cells.Several factors influence the antigenicity of tumors evolving in immunocompetent, syngeneic hosts. First, the mutational burden  is heterogeneous across and within tumors, ranging from ~1 mutation/Mb in hematologic malignancies to >10 mutations/Mb in solid tumors with a hypermutant phenotype.Sources of ICD adjuvanticity), at least some ICD-triggering regimens may also boost antigenicity. This applies to potentially mutagenic agents, and to interventions that drive the reactivation of endogenous retroviruses and/or induce the expression of mutated genes or TAAs, such as CDK4/CDK6 inhibitors,Of note, while the majority of ICD inducers are believed to have little impact on antigenicity and to operate by driving the correct spatiotemporal emission of DAMPs in the context of cell death presentation to naïve T cells. MAMPs include microbial nucleic acid species  as well as structural components , and mostly mediate immunostimulatory effects via pattern recognition receptors (PRRs).The same PRRs activated by MAMPs are also involved in the adjuvanticity of ICD induced in cancer cells by non-microbial stimuli.Macroautophagy (herein referred to as autophagy) is a cytoprotective lysosomal pathway for the degradation of superfluous or potentially dangerous cytosolic material and organelles.2+ levels,The integrated stress response (ISR), a multipronged molecular mechanism for the preservation of cellular homeostasis is required for the exposure of ER chaperones on the cell surface during ICD.The mechanisms for the preservation of cellular homeostasis in response to infection are also intimately involved in the adjuvanticity of ICD, even when the latter is not driven by microbes.The precise stress responses involved in the release of other ICD-associated DAMPs including ANXA1 and HMGB1 remain to be fully elucidated. Irrespective of this unknown, ANXA1 is known to spatially direct APCs to dying cells on interaction with formyl peptide receptor 1 (FPR1),-/-Fpr1 mice).P2R×7, TLR4 and FPR1 polymorphisms in patients with breast carcinoma receiving neoadjuvant anthracyclines)The critical role of DAMP signaling in the immunogenicity of RCD has been established by a plethora of complementary mechanistic experiments aimed at: (1) the pharmacological or genetic inhibition of the signaling pathways involved in DAMP release In vivo models).Although some tissues respond to pathogenic infection more robustly than others , cells succumbing to microbial infection generally drive adaptive immunity irrespective of anatomical location.There are several mechanisms whereby the microenvironment of developing tumors can antagonize the initiation or execution of ICD, largely reflecting the ability of various neoplasms to establish peripheral tolerance. So-called ‘cold’ and ‘excluded’ tumors are poorly infiltrated by immune cells including APCs and their precursors at baseline, implying that the likelihood for dying cancer cells and their corpses to be productively processed and drive cross-priming is reduced.+CD25+FOXP3+ regulatory T (TREG) cells, M2-polarized tumor-associated macrophages (TAMs), and/or myeloid-derived suppressor cells (MDSCs).REG cells, M2-polarized TAMs and MDSCs also have direct ICD antagonizing effects.Moreover, the activity of APCs that infiltrate malignant lesions is generally inhibited by immunosuppressive cytokines including (but not limited to) IL-10 and transforming growth factor beta 1 (TGFB1).The redox status of the TME and individual DAMPs or their receptors may also affect the ability of RCD to drive adaptive anticancer immunity. For example, the release of oxidized HMGB1 by cancer cells undergoing pyroptosis, a gasdermin-dependent form of RCD generally associated with inflammasome activation,Another major mechanism for progressing tumors to evade ICD at the execution phase  relies on immune exhaustion, that is, the establishment of dysfunction in tumor-infiltrating T cells.Importantly, multiple ICD-inducing therapeutics as well as various therapies that do not promote ICD can mediate immunomodulatory effects on the TME by directly interacting with immune cell populations (rather than with cancer cells).Over the past two decades, an intensive wave of investigation has unveiled major mechanistic and correlative aspects of ICD as a process culminating in the activation of adaptive immunity against dying cells. Experimental strategies conceived to assess the immunogenicity of RCD encompass: (1) the study of DAMP emission from (and activation of relevant stress responses in) dying cells; (2) biochemical and functional tests to assess the activation of APCs and their ability to mediate cross-priming, in vitro and (3) the ability of dying cells to initiate adaptive immunity in vivo, in immunocompetent syngeneic hosts. Here, we will summarize current methods to assess ICD in oncological settings.Robust experimental evidence indicates that the ability of RCD to promote adaptive immunity critically relies on a progressing (but not exhausted) CTL response.Intracellular responses to stress mechanistically involved in DAMP release have also been employed as surrogate ICD biomarkers. ISR activation has generally been monitored in its three reticular components by the assessment of eIF2α phosphorylation, X-box binding protein 1 (XBP1) splicing and activating transcription factor 6 (ATF6) nuclear translocation, in some instances along with heat shock protein family A (Hsp70) member 5  upregulation. These biomarkers are detected by immunoblotting, flow cytometry, immunofluorescence microscopy, immunohistochemistry and/or RT-PCR, using dedicated antibodies or probes.Cell surface exposure of ER chaperones as a surrogate biomarker of ICD can be determined by (1) flow cytometry in non-permeabilized early apoptotic cells using specific antibodies or dedicated constructs  combined with vital dyes like 7-aminoactinomycin D (7-AAD), 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI), which enable the exclusion of dead cells from the analysisTwo main approaches have been harnessed to determine the release of ICD-associated soluble DAMPs and cytokine from dying cells: (1) DAMP/cytokine quantification in culture supernatants and (2) quantification of residual DAMPs/cytokines in the intracellular microenvironment. Of note, while the former approach is universally applicable, the latter cannot be employed for DAMPs and cytokines that are actively synthesized before release, such as type I IFN and CXCL10.In vivo models). To partially circumvent this issue and enable a functional assessment of the immunogenicity of RCD in both the mouse and human system, the field has borrowed multiple classical assays from immunology. In general, these experiments aim at evaluating whether dying cancer cells can stimulate the ability of APCs to optimally cross-prime CTLs and hence initiate an adaptive immune response.While intracellular responses to stress and DAMP emission can be equally monitored in mouse and human tumor models, in vivo studies can currently be performed only in the former  their ability to engulf dying cells or their corpses; (2) their maturation status and migratory capacity and (3) their ability to cross-present antigenic material to CTLs. Phagocytosis is often assessed by coculturing APCs or their precursors and dying cancer cells on individual prelabeling of both compartments with distinct non-toxic fluorescent dyes that remain in the cytoplasm, such as carboxyfluorescein succinimidyl ester (CFSE) or PKH26, followed by fluorescence microscopy or flow cytometry.The cross-priming potential of APCs exposed to cancer cells undergoing RCD has most frequently been assessed by coculturing them with syngeneic, naïve T cells, followed by the assessment of: (1) proliferative T cell response, generally by flow cytometry on previous labeling with CFSEs.c., in the absence of any immunological adjuvant and on the removal of exogenous chemical entities . One to two weeks later, mice are challenged s.c. with living cancer cells of the same type  and followed over 40–60 days for tumor incidence and growth.+ T cells cross-primed in vitro by APCs exposed to cancer cells undergoing ICD have been employed in adoptive transfer experiments to treat tumors previously established with the living cancer cells of the same type.Interpretation ofICD).Only a few models are currently available to investigate ICD in vivo . The gols.c., at anatomically distant sites or (2) metastasis-prone cancer cells to generate a palpable lesion s.c. and metastatic (lung) dissemination.Three alternative approaches to assess ICD in vivo, in immunocompetent syngeneic systems, critically rely on the measurement of tumor growth at non-treated disease sites, which implies they can only be implemented with localized therapies All these models are amenable to ex vivo studies aimed at: (1) the characterization of DAMPs released by cancer cells responding to stress in situ; (2) the immunological profiling of APCs and CTLs underlying the initiation and execution of anticancer immunity in vivo and (3) the identification of mechanistic vs correlative aspects of ICD elicited in vivo by the stressor(s) of choice .Most of the assays that are currently available to estimate the propensity of RCD to drive adaptive immune responses fail to take into proper consideration the complexity that is inherent to ICD, which (as detailed above) ultimately represents a highly contextual process that depends on (1) initiating stimulus, (2) responding cell and (3) the host . In thisSurrogate biomarkers of ICD, such as the accumulation of cytosolic DNA, the exposure of ER chaperones on the cell surface or the release of ATP and/or HMGB1 by dying cells, as well as biomarkers of the stress responses that underlie their emission, have been instrumental for the characterization of the key molecular players of process, and the identification of potential ICD inducers in large screening efforts.Although their scalability to screening applications is limited, immunological assays testing the ability of APCs primed with dying cells to engage in the sequential process leading to cross-priming (which is an absolute requirement for adaptive immunity in this setting) obviously offer a more precise assessment of the immunogenicity of RCD. However, these assays are also inherently limited in that they are unable to assess two major prerequisites for cross-priming: (1) the ability of APCs to physically reach sites of RCD and (2) the existence of naïve T cell clones specific for antigens expressed by dying cells, ultimately calling for validation with in vivo models.Vaccination assays have been highly instrumental for the demonstration that syngeneic dying cells can drive adaptive immunity in the presence of adequate antigenicity and adjuvanticity.As a group, ICD-relevant in vivo models are affected by multiple issues including the fact that only murine systems can be investigated, and it cannot be excluded that the molecules and cell populations at play in the human setting may be different (at least to some degree).Current efforts to circumvent, at least in part, these issues involve the use of orthotopic models , transgene-driven models (which may offer a superior view on early oncogenesis) and carcinogen-driven models (which have superior heterogeneity).In summary, the assessment of ICD requires experimental support from a variety of in vitro and in vivo assays that should cumulatively assess the ability of malignant cells undergoing RCD at the natural anatomical location to recruit APCs and stimulate them to initiate adaptive anticancer immunity.Accumulating evidence demonstrates that the initiation of ICD is critical for the elimination of infectious pathogens and stands out as a major therapeutic goal for cancer therapy, especially in consideration of the current clinical success of ICBs.2, and adenosine, especially in the context of ICD.There are three major obstacles precluding the full clinical potential of ICD inducers to be realized. First, most of these agents have been developed clinically based on the paradigm of maximum tolerated dose (MTD), and it is now clear that greater cytotoxicity does not necessarily coincide with optimal immunogenicity.Along with the establishment of humanized rodent models that enable the investigation of ICD in vivo , we believe that these issues currently stand as the major challenges for the field in the near future. We surmise that the clinical efficacy of numerous agents currently employed for the management of cancer could be remarkably boosted if we acquire the capacity to use them as ICD inducers. Novel technologies are constantly improving our ability to monitor the immunological changes occurring in patients responding to ICD inducers, including alterations in the intratumoral and circulating TCR repertoire.