Inappropriate antifungal use is prevalent and can lead to drug-resistant fungi, expose patients to adverse drug events, and increase healthcare costs. While antimicrobial stewardship programs have traditionally focused on antibiotic use, the need for targeted antifungal stewardship (AFS) intervention has garnered interest in recent years. Despite this, data on AFS in immunocompromised patient populations is limited. This paper will review the current state of AFS in this complex population and explore opportunities for multidisciplinary collaboration. Antimicrobial Stewardship Programs (ASPs) optimize patient outcomes through coordinated efforts aimed at improving antimicrobial prescribing ,2,3,4. IWhile the impact of ASPs on antibiotic use has a robust body of literature, the impact of AFS programs, particularly among immunocompromised patients, is less described. The assessment of antifungal use in this population requires an understanding of host factors that predispose patients to invasive fungal infections (IFIs) and appropriate prophylaxis and management strategies. Additionally, limitations of diagnostic testing and an understanding of the complex pharmacological properties of antifungals must be considered when constructing AFS interventions. This paper will highlight the current state of AFS in immunocompromised patients, describe the limitations of traditional and non-culture-based fungal diagnostics, and highlight the importance of a multidisciplinary team with expertise in caring for patients requiring antifungals.Many of the principles of antibiotic stewardship can be incorporated into AFS programs. In fact, many of the AFS core elements proposed by the MSGERC  mirror tTools commonly used in general ASP interventions such as prospective audit and feedback and preauthorization may be leveraged to improve antifungal prescribing. Prospective audit and feedback engages the provider after an antibiotic is prescribed whereas preauthorization requires approval before an antimicrobial can be initiated. The advantages and disadvantages of both strategies have been described previously . FinallyStaphylococcus aureus bacteremia and has been linked to improved patient outcomes [Published AFS interventions have demonstrated reductions in antifungal consumption and antifungal drug expenditure without adversely affecting clinical outcomes . Recent outcomes ,21. Whiloutcomes .Despite this promising data, caution should be exercised when attempting to generalize these findings to immunocompromised populations. Non-candida opportunistic yeasts, many of which are echinocandin non-susceptible, may disproportionately affect immunocompromised patients and make the application of these bundles challenging ,23,24,25IFIs are associated with a high rate of morbidity and mortality in hematology-oncology patients . As a reA recent study in pediatric hematology-oncology patients evaluated antifungal prescribing before and after implementation of a protocol for the management of IFIs . After iA study in adult hematology patients evaluated the impact of a multi-pronged stewardship intervention over nine years . The intGuidelines for hematology-oncology patients should also be developed to address considerations for initiation of antifungal therapy, diagnostic work-up, and recommendations for consultation with ID physicians. These algorithms should incorporate evidence-based recommendations from national practice guidelines and provide options based on the availability of formulary antifungals. Education could then be provided to hematology-oncology clinicians to support guideline implementation. Prospective audit and feedback, an intervention associated with improved long-term acceptance rates, can then be used to provide clinicians feedback and assess guideline compliance .Institutional guidelines can also be used to address high-risk hematology-oncology patients in whom antifungal prophylaxis has been associated with clinical benefit  patients receiving high-intensity induction chemotherapy) and discourage use in lower-risk patients where no clear benefit has been established . These gIFIs in SOT patients are an important cause of morbidity and mortality . This, cBuilding on the concept of an ASP “timeout” which has been evaluated for ASPs and supported by the CDC , MularonMany of the traditional fungal diagnostics are neither sensitive nor specific for IFIs and make establishing a definitive diagnosis challenging ,43,44. Hd-glucan (BDG), galactomannan, Aspergillus polymerase chain reaction (PCR), mannan, and antimannan [Several non-culture-based tests (NCBT) are used clinically in the diagnosis of IFIs. These include (1→3)-β-timannan ,47. Alontimannan ,49. The timannan ,51. Whiltimannan . While fDespite their promising role in the diagnosis of IFIs, the lack of specificity of NCBTs may drive unnecessary antifungal use. In a study of non-neutropenic patients who were ordered a BDG at an academic medical center, a retrospective review by 2 independent ID physicians revealed that orders were inappropriate in nearly 50% of cases . SimilarIn addition to specialty physicians, clinical pharmacists are critical allies in AFS efforts .In one study, contraindicated DDIs were reported in nearly 25% of inpatients prescribed mold-active triazoles . Many ofMany of the antifungals used for the treatment and prevention of IFIs also have complex pharmacokinetics that leads to inter-and intra-patient variability in drug exposure ,60. ThisClinical microbiologists are key collaborators of a robust AFS program. In addition to helping with the implementation and interpretation of traditional and NCBTs, they are critical in the validation of antifungal susceptibility testing (AST). They are uniquely aware of the limitations of AST and may be the only members of the team with in-depth knowledge of interlaboratory variability among testing platforms or methodologies.Laboratory collaboration is also key to ensure the timely in-house availability of antifungal drug levels as these have been associated with reduced time to drug concentration results and time to therapeutic serum concentrations . In collAlthough limited data exist regarding effective AFS interventions in immunocompromised patients, published strategies to date are reassuring and should motivate future research in this space. It is not surprising that many of these interventions mirror principles of general ASPs . When the limited number of antifungal drugs available is considered, the argument for expanded AFS is compelling. As new antifungal drugs will be approved in the coming years, the need for a dedicated team of clinicians to steward their use will only increase . Despite


In this review, we will present the different roles of stromal cells in pancreatic cancer, focusing on CAF origin as a source of heterogeneity, and the role this may play in therapy failure. We will discuss preclinical models that could be of benefit to the field and that may contribute to further clinical development.Pancreatic tumors are known to harbor an abundant and highly desmoplastic stroma. Among the various cell types that reside within tumor stroma, cancer-associated fibroblasts (CAFs) have gained a lot of attention in the cancer field due to their contributions to carcinogenesis and tumor architecture. These cells are not a homogeneous population, but have been shown to have different origins, phenotypes, and contributions. In pancreatic tumors, CAFs generally emerge through the activation and/or recruitment of various cell types, most notably resident fibroblasts, pancreatic stellate cells (PSCs), and tumor-infiltrating mesenchymal stem cells (MSCs). In recent years, single cell transcriptomic studies allowed the identification of distinct CAF populations in pancreatic tumors. Nonetheless, the exact sources and functions of those different CAF phenotypes remain to be fully understood. Considering the importance of stromal cells in pancreatic cancer, many novel approaches have aimed at targeting the stroma but current stroma-targeting therapies have yielded subpar results, which may be attributed to heterogeneity in the fibroblast population. Thus, fully understanding the roles of different subsets of CAFs within the stroma, and the cellular dynamics at play that contribute to heterogeneity in CAF subsets may be essential for the design of novel therapies and improving clinical outcomes. Fortunately, recent advances in technologies such as microfluidics and bio-printing have made it possible to establish more advanced  The tumor microenvironment (TME) comprises both cellular and non-cellular components . It consInitially, CAFs were assumed to be a homogeneous population of stromal cells. However, various studies have revealed heterogeneity within the CAF pool and identified that these cells can possess both pro- and anti-tumorigenic properties . Indeed,In context of pancreatic cancer, CAFs commonly derive from three major sources: resident fibroblasts , pancreaFibroblasts are mesenchymal cells with essential roles throughout embryonic development as well as adult organ function where they provide mechanical support and maintain tissue architecture . During Hepatic stellate cells were originally identified by Mesenchymal stem cells are multipotent adult progenitor cells that were initially discovered in the bone marrow but were later documented in multiple other tissues, including umbilical cord and fat tissue . MesenchBesides the abovementioned sources, CAFs have been documented to transdifferentiate from seemingly unrelated cell types such as epithelial cells , endotheIn a recent time-course scRNA-seq study, fibroblasts from pre-invasive human and mouse pancreatic lesions were analyzed to shed some light on gradual changes during pancreatic tumorigenesis and possibly the origin of such cells . A mouseAnother single cell transcriptomic study also investigated the biology of primary PDAC tumors as well as metastatic lesions from human patients . A notewin situ hybridization) and IF (immunofluorescence) on human PDAC sections with different clinical stages. The findings of this study suggested that csCAFs may play a tumor-suppressive role in PDAC microenvironment and that this population is gradually lost during tumor progression.A recent scRNA-seq paper by Another prominent study described a mass cytometry approach that allowed the analysis of mesenchymal stroma in both normal and tumor murine pancreatic tissues . The finorigin is still somewhat limited. This underscores the need for more comprehensive approaches that make good use of scRNA-Seq findings, perhaps together with lineage tracing technologies to better understand how tumor stroma is shaped and identify the contributions and roles of different CAF progenitor cells in PDAC tumors. A recent paper highlights the importance of such tracing studies for understanding PDAC; targeted ablation specifically of PSC-derived CAFs revealed non-redundant functions for this defined CAF population in shaping the PDAC microenvironment , platelet-derived growth factor (PDGF), and fibroblast growth factor 2 (FGF2) are considered key regulators of fibroblast recruitment and activation . In pancThe SHH pathway is an important signaling axis in PDAC as it is involved in pancreatic fibrogenesis . InitialEpigenetic regulation of normal fibroblasts has been recently highlighted as a means of conversion into CAFs in different cancers . Mechanistically, exposure of the fibroblasts to pro-inflammatory leukemia inhibitory factor (LIF) triggers an epigenetic switch leading to constitutive activation of Janus kinase 1–signal transducer and activator of transcription 3 (JAK1–STAT3) signaling, which is sustained by the DNA methyltransferase DNMT1, and activates the fibroblasts into pro-invasive CAFs with increased acto-myosin contractility ; this suexosomes below) to educate members of the TME and contribute to carcinogenesis  signaling and high levels of the NF-κB target gene tumor necrosis factor-α (TNF-α) . AnotherIn conclusion, we highlighted how pancreatic cancer cells shape the TME by recruiting and educating CAFs from different sources/precursor cells and presented the main signaling pathways that are involved in these phenomena and how they dictate CAF subtype. We also went further and stressed the importance of cancer cell genotype in further increasing heterogeneity in the CAF pool and generating CAFs that encourage PDAC cell aggressiveness and support tumor progression.Cancer-associated fibroblasts are a substantial source of signals that can promote tumor growth and impact on therapy responses . CAFs caAnother method by which fibroblasts interact with tumor cells is the exchange of metabolites. Many recent studies have focused on metabolites and their importance in tumor-stroma interaction. Here, we will briefly mention some examples of how stromal cells support pancreatic cancer growth by providing metabolites. For instance, fibroblasts are known for undergoing autophagy in response to stimulation by cancer cells and thereby releasing alanine, which subsequently contributes to pancreatic cancer cell energy production by feeding the tricarboxylic acid (TCA) cycle . PSC-deracquired pancreatic cancer resistance. Many efforts aimed to explore the secretome of CAFs and various secreted factors and active substances have been identified to be involved in tumor-stroma crosstalk and might serve useful to better understand CAF heterogeneity and establish effective treatment approaches.Many recent studies have been aimed at studying the role of extracellular vesicles, such as exosomes, in tumor-stroma interaction . Exosomein vitro findings and whether targeted approaches could be safe and efficacious in the clinic.The notion of a tumor-suppressive population of CAFs has gained a lot of attention. This led to the proposal of markers, such as α-smooth muscle actin (SMA), as candidates to identify tumor-restraining CAFs in PDAC. However, other studies had already revealed a correlation between the number of α-SMA+ CAFs and poor outcome in various types of human solid cancers, leading to an opposing hypothesis which states that α-SMA+ CAFs are in fact tumor-permissive and tumor-promoting . InteresThere, thus, appears to be a link between CAFs expressing markers that are typically attributed to MSCs and/or PSCs and them being tumor-repressive. However, since MSCs and PSCs are major sources for CAFs, this suggests one of two scenarios; (i) that tumor-repressive CAFs are less activated and therefore more similar to MSCs/PSCs, or (ii) that there is heterogeneity among MSCs/PSCs that persists after they become CAFs. These notions seem less farfetched when you take into account the findings of Exosomes are also involved in mediating the anti-cancer effects of stromal cells in pancreatic cancer. One study demonstrated that BMSC-derived exosomes could suppress proliferation, invasion, and metastasis as well as promote apoptosis in pancreatic cancer cells by transferring miR-126-3p and, thereby, downregulating ADAM9 . Anotherex vivo or animal models should be utilized in order to corroborate these findings and make way to clinically relevant targeted approaches that target specific CAF subpopulations in PDAC. We will discuss this in a subsequent section that discusses available technologies and how they may be utilized for improving PDAC modeling.To sum up, we know that certain aspects dictate whether CAFs exert tumor-suppressive or -promoting activities and that this needs to be fully understood in order to improve therapeutic regimens and/or adopt more targeted approaches. Taking the abovementioned information into account, there seems to be a link between the tumor-suppressive activity of CAFs and them expressing PSC/MSC markers and that tumor-suppressive CAFs seems to have reduced α-SMA expression and ECM remodeling abilities compared to their tumor-promoting counterparts. We propose that more advanced in vivo setting. USP27X is another interesting candidate in context of stroma-mediated chemoresistance. In fact, USP27X is activated by TGFβ, a known inducer of EMT  . This isRadiation therapy has a prominent place in treating locally advanced pancreatic cancer . UnfortuA recent study described the role of stromal fibrosis in activating pro-survival and epithelial-to-mesenchymal transition (EMT) pathways in PDAC. The group identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT) and suggested that activation by ephrinB2 drives fibroblasts toward a myofibroblast differentiation, thereby driving cancer invasion . UnfortuAs was introduced above, exosome transfer is a prominent mechanism for tumor-stroma signaling. This mode of communication is also implicated in conferring resistance to radiation . For insAnother study highlighted the important role of tumor stroma in hampering or even negating the beneficial effects of radiotherapy in PDAC treatment . SpecifiCancer-associated fibroblasts and bone marrow cells have been noted to protect breast cancer cells by inducing interferon (IFN)-related DNA damage-resistance in a STAT1-dependent manner, leading to radio-resistance . AlthougSimilarly, conditioned medium from PSCs led to a dose-dependent induction of pancreatic cancer cell proliferation, migration, invasion, and colony formation and caused resistance to gemcitabine and RT . The mecStromal cell-mediated radioresistance can be also induced through direct contact-mediated signaling. In fact, PSCs promote radioprotection and stimulate the proliferation of pancreatic cancer cells through β1 integrin signaling, which is known to modulate genotoxic stress-induced cellular responses such as RT . NotablyConsidering the relevance of RAS signaling in PDAC, it would be interesting to better understand how this pathway facilitates treatment resistance in this disease and how this could be exploited in the clinic. Indeed, inhibiting Ras activation could be a potential strategy for tumor-specific radiosensitization in a large majority of pancreatic cancer patients . AnotherVarious studies have demonstrated the complex contributions of immune cells to pancreatic cancer, and how they communicate with cancer cells as well as other members of the TME . An impoMyofibroblasts are perhaps the most studied stromal cells in PDAC and the contributions of fibroblasts falling under the myCAF phenotype have been investigated from different angles with respect to PDAC progression and therapy response/resistance. Inhibition of TGF-β, a promoter of the myCAF phenotype, has gained some attention in the clinic and has been successfully utilized in concert with gemcitabine to improve overall survival in unresectable PDAC patients . InteresAs outlined earlier, SHH signaling is important to consider in PDAC also from an immunological perspective. Initially, it was shown that depletion of CAFs could induce immunosuppression in PDAC tumors with indications that the Hedgehog pathway may play a role in this phenomenon . This isAnother study also explored the premise of altering the fibroblast composition as a therapeutic strategy for PDAC. It was identified that tumor-secreted IL1, predominantly through autocrine LIF, activates the JAK/STAT pathway in CAFs . SubsequTogether, the abovementioned studies introduce some of the pathways by which CAFs are involved in response to immunotherapy and underline the importance of targeting select CAF subsets or certain pathways that underpin resistance to immunotherapy rather than using general stromal disruptive agents or targeting the entire CAF population.MRC2) receptor that supported tumor growth and metastasis  transcriptional factor is a key regulator of epithelial-to-mesenchymal transition (EMT) and metastatic colonization in PDAC. Prrx1 is also highly expressed in PDAC stroma and was reported to mediate CAF activation, leading to increased ECM deposition, improved tumor differentiation, fewer circulating tumor cells, and reduced metastasis . CAFs exBesides, characterizing stroma within metastatic lesions in an autochthonous model of PDAC indicated that myofibroblasts appear when metastases are as small as 6–7 cells and that cell populations within these lesions become more epithelial during growth . InteresOne paper eloquently introduces the processes involved in the metastatic dissemination of pancreatic cancer cells . It was Together, the abovementioned studies highlight the importance of CAFs in pancreatic cancer metastasis and illustrate the need for more comprehensive studies on the cellular origins of CAFs or MAFs in PDAC metastases and how they phenotypically differ from their counterparts in primary sites.Another aspect that is important to consider is treatment-induced changes to the TME. During the process of being activated, and in response to therapeutic regimens, CAFs undergo changes that grant them resistant characteristics; this mainly occurs through a defective p53/p21 response pathway and high expression of the cancer marker Survivin . Since tCancer-associated fibroblasts and mesenchymal stem cells have been consistently shown to be enriched in chemotherapy-treated human tumors, including PDAC, wherein they promote cancer growth and treatment resistance by secreting various paracrine factors . Not onl+) chemokines. On the contrary, administering the same overall dose of a certain drug over a longer time frame, as a low-dose metronomic chemotherapy regimen, largely reduced therapy-induced stromal ELR+ chemokine paracrine signaling and, thus, enhanced treatment response and improved mouse survival rates  cell culture systems have been used to study and understand human pathology. However, despite the fact that these models led to various scientific advances, their utility for modeling human physiology and pathology is limited for several reasons . Hence, ex-vivo culture systems is a critical step to establish a platform for the study of immunotherapy in pancreatic cancer  be able to recapitulate the phenotypes and functionality of different CAF populations and subsets in PDAC tumors. Of course, this is essential to allow proper drug response predictions as the stroma is one of the main obstacles for drug delivery as well as an important contributor to resistance in pancreatic cancer.Taken together, the abovementioned tools present good possibilities for modeling PDAC. Considering the dynamic and heterogeneous nature of PDAC tumors, it is essential to use advanced models that allow for co-culturing cancer cells with different members of the stroma in order to recreate the ex vivo models need to be implemented in combination with lineage tracing and scRNA-seq technology. This combination might not be currently practical or feasible in light of technological limitations; however, this does not reduce from value of establishing and implementation such platforms in the near future. In conclusion, we believe that accurately modeling PDAC and unraveling the paradigm that is CAF heterogeneity are crucial for reaching clinically relevant findings and making strides toward personalized or targeted treatment approaches.It has become apparent throughout this paper how important CAFs are in PDAC tumors and how complex and seemingly opposing their contributions can be. It is for this reason that we would like to stress the urgency of more advanced studies that focus on the cellular origins of CAFs, how these precursors contribute to the CAF population, and how this drives stromal heterogeneity in PDAC. We are also in need of markers that can be used to accurately identify different CAF subsets and their potential function, possibly as potential biomarkers to select for (novel) targeted therapeutic approaches. It might, thus, be relevant to investigate and better understand the gradual variations in the stromal profile during PDAC progression. In order to make headway on this matter, we believe that more advanced PM drafted the manuscript and figures. HL and MB provided supervision and revised the manuscript. All authors have read and accepted the final version of the manuscript.PM has nothing to disclose. MB has received research funding from Celgene and reports an advisory role for Servier outside the scope of the submitted work. HL reports grants from Bristol-Myers Squibb, grants from Bayer Schering Pharma, grants from Celgene, grants from Janssen-Cilag, grants from Lilly, grants from Nordic Group, grants from Philips Healthcare, grants from Roche, grants from Merck Sharp and Dohme, personal fees from Lilly, personal fees from AstraZeneca, personal fees from Lilly, personal fees from Nordic, personal fees from Bristol-Myers Squibb, outside the scope of the submitted work.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We conclude that implementation of feasible dietary approaches holds the promise to attenuate the burden of cardiovascular disease and facilitate healthy aging in humans.The average human life expectancy continues to rise globally and so does the prevalence and absolute burden of cardiovascular disease. Dietary restriction promotes longevity and improves various cardiovascular risk factors, including hypertension, obesity, diabetes mellitus, and metabolic syndrome. However, low adherence to caloric restriction renders this stringent dietary intervention challenging to adopt as a standard practice for cardiovascular disease prevention. Hence, alternative eating patterns and strategies that recapitulate the salutary benefits of caloric restriction are under intense investigation. Here, we first provide an overview of alternative interventions, including intermittent fasting, alternate-day fasting and the Mediterranean diet, along with their cardiometabolic effects in animal models and humans. We then present emerging pharmacological alternatives, including spermidine, NAD Cardiovascular diseases remain the major cause of morbidity and mortality, accounting for 17.9 million deaths per year or almost one third of all deaths worldwideExtensive body of evidence indicates that hypertension is the leading modifiable risk factor for cardiovascular disease and premature mortality , accountIn general, patients with cardiovascular disease are commonly affected by more than one risk factor . EmerginThe amount of dietary intake, quality of food and its preparation as well as micronutrient composition together with general eating habits  significantly contribute to the onset of cardiovascular disease risk factors , 10. To The Mediterranean diet is characterized by high fruit and vegetable intake combined with plenty of fish and unsaturated fatty acids derived mainly from extra-virgin olive oil, with minimal or no consumption of low saturated fat and processed food. Many epidemiological studies and randomized clinical trials report that the traditional Mediterranean diet is associated with lower risk for all-cause and cardiovascular disease mortality, coronary heart disease, metabolic syndrome, and diabetes mellitus type 2 , 38. ForGrowing evidence suggests that the markedly reduced risk for cardiovascular disease by the Mediterranean diet is attributed to its plant-rich nutrient composition with seafood as the predominant source of animal protein. For example, the Women's Health Initiative Observational Study demonstrated that increased consumption of baked or boiled fish, but not fried fish, inversely correlates with heart failure risk in postmenopausal women . In agreCaloric restriction and other forms of stringent eating behaviors, such as intermittent fasting and alternate-day fasting, have recently attracted a lot of attention amongst researchers and have become increasingly popular in the general population to avoid the unhealthy effects of “all-around-the-clock” high caloric diet. Caloric restriction is defined as a chronic reduction of overall calorie consumption without malnutrition. In patients with metabolic syndrome, caloric restriction reduces body weight and exerts beneficial effects on insulin levels, fasting glucose levels, lipoprotein composition and pro-inflammatory cytokines within 6 months of intervention . In addiCaloric restriction was shown to be a safe and well-tolerable intervention in healthy, non-obese individuals , leadingMechanistically, the beneficial effects of caloric restriction are closely linked to autophagy, a cellular recycling process essential for cardiovascular homeostasis , 45. CalNotably, severe caloric restriction  induces changes of gut microbiome composition during weight loss , 51. HowCollectively, caloric restriction exerts clear cardiometabolic benefits in both obese and non-obese individuals. However, caloric restriction might also cause adverse side effects on immunity, fertility and bone density. Hence, further research is warranted to develop more suitable dietary patterns or pharmacological alternatives to reproduce the health benefits of caloric restriction.ad-libitum food intake for 24 h are also practiced and known as alternate-day fasting. Although intermittent fasting and alternate-day fasting are not as well-studied as caloric restriction, emerging evidence suggests that they are more tolerable and their side effects are less prominent than in caloric restriction and, thus, both dietary interventions could represent promising and more feasible strategies to curtail the hypercaloric pandemic in the Western societies  in obese diabetic patients at risk of cardiovascular disease showed that both regimens reduce body weight and HbA1c levels, a measure of long-term blood glucose control . ConsistWith regard to alternate-day fasting, a short-term trial conducted in obese adults, which showed high adherence to alternate-day fasting at least for 8 weeks, revealed manifold cardiometabolic benefits, including reduced body weight, body fat percentage, total and LDL-cholesterol, triglycerides as well as systolic blood pressure . It is iAlong similar lines, initial short-term studies in non-obese individuals highlighted the positive impact of alternate-day fasting on body weight loss in absence of clear metabolic changes, but increased fat oxidation. Notably, participants reported difficulty to adhere to alternate-day fasting due to severe hunger on the fasting days . By contIn sum, growing body of evidence indicates potential cardiovascular benefits of intermittent and alternate-day fasting . HoweverRecent years have seen an increasing interest in fasting-mimicking diets and caloric restriction, which might offer a more feasible alternative to stringent forms of fasting. For example, a randomized clinical trial was designed to investigate the effects of fasting mimicking diets, which are low in carbohydrates and protein and high in unsaturated fats, on cardiovascular disease and risk factors, including aging and diabetes mellitus type 2 . The autDahl rats, while reducing maladaptive hypertrophy and attenuating the decline of diastolic function  and nicotinamide mononucleotide (NMN), has been shown to alleviate metabolic abnormalities by reducing body weight gain and reinstating blood glucose control in mice consuming HFD  clinical trialRecent years have seen a growing interest in understanding how dietary interventions shape and interact with the most common cardiovascular risk factors, including hypertension, obesity, metabolic syndrome, and diabetes mellitus type 2. Substantial cardiometabolic improvements have been reported with fasting interventions such as reduction in blood pressure, body weight and fat mass, lower blood glucose, and improvement in insulin sensitivity, both in experimental and clinical studies. Although caloric restriction consistently improves several aspects of health, its application has been hampered by poor compliance and adverse side effects on bone health and immune response, especially in the elderly. To overcome these major hurdles, clinical trials on alternate-day or intermittent fasting, with higher statistical power and follow-up, are strongly needed before they can be implemented as a treatment strategy. Individuals practicing alternate-day or intermittent fasting should consider to include regular physical activity to maintain their energy expenditure. Emerging evidence indicates that the optimal cardioprotective diet is constructed around the traditional Mediterranean eating pattern.Another interesting aspect that warrants further attention is the effect of caloric restriction mimetics or dietary interventions aimed at weight loss on the gut microbiome changes in obese patients with diabetes mellitus type 2 or metabolic syndrome. Although these interventions propose beneficial clinical outcomes, their effect on the gut microbiome is only beginning to unfold. Interestingly, a combination therapy of resveratrol and spermidine synergistically induces autophagy at doses, which do not trigger effects of the same magnitude if administered alone. At present, however, it remains elusive what is the optimal dose for any of the caloric restriction mimetics that could provide health benefits or protect humans at risk of cardiovascular disease.Unlike the current drug development approaches that focus on individual diseases in isolation and consider specificity as a desirable outcome in disease prevention and treatment, both caloric restriction mimetics and caloric restriction exhibit a spurious mode of action, intercepting with multiple different targets . Such plNon-cell autonomous effects of caloric restriction mimetics and caloric restriction itself, such as the anti-inflammatory or immune modulatory functions, are increasingly viewed as relevant as cell autonomous mechanisms. Taking this into account, more research is needed to ascertain how different forms of fasting and caloric restriction mimetics can be the most favorable to further improve cardiometabolic markers in healthy adults and patients living with or at risk of developing cardiovascular disease. Based on the currently available data, harnessing caloric restriction mimetics or dietary interventions, such as intermittent fasting or the Mediterranean diet represent a promising preventive venue, which might reduce cardiovascular risk and the burden of cardiovascular disease.SS conceptualized the manuscript. JV, MA, and SS contributed to the research for writing the manuscript. JV and SL-H designed the figures and table. All authors contributed to the discussion, writing, and review of the manuscript.This work was supported by the Austrian Science Fund–FWF (I3301) and the European Research Area Network on Cardiovascular disease  to SS. MA acknowledges funding received from the European Society of Cardiology, the Austrian Society of Cardiology (Präsidentenstipendium der ÖKG), and the Medical University of Graz (START Fund). SL-H reports funding by the Austrian Science Fund - FWF (V530) and BioTechMed-Graz (Young Researcher Groups [YRG]).MA and SS are involved in a patent application related to the cardiometabolic effects of caloric restriction mimetics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite advances in modern medicine, brain tumor patients are still monitored purely by clinical evaluation and imaging. Traditionally, invasive strategies such as open or stereotactic biopsies have been used to confirm the etiology of clinical and imaging changes. Liquid biopsies can enable physicians to noninvasively analyze the evolution of a tumor and a patient’s response to specific treatments. However, as a consequence of biology and the current limitations in detection methods, no blood or cerebrospinal fluid (CSF) brain tumor-derived biomarkers are used in routine clinical practice. Enhancing the presence of tumor biomarkers in blood and CSF via brain-blood barrier (BBB) disruption with MRI-guided focused ultrasound (MRgFUS) is a very compelling strategy for future management of brain tumor patients.A literature review on MRgFUS-enabled brain tumor liquid biopsy was performed using Medline/Pubmed databases and clinical trial registries.The therapeutic applications of MRgFUS to target brain tumors have been under intense investigation. At high-intensity, MRgFUS can ablate brain tumors and target tissues, which needs to be balanced with the increased risk for damage to surrounding normal structures. At lower-intensity and pulsed-frequency, MRgFUS may be able to disrupt the BBB transiently. Thus, while facilitating intratumoral or parenchymal access to standard or novel therapeutics, BBB disruption with MRgFUS has opened the possibility of enhanced detection of brain tumor-derived biomarkers.In this review, we describe the concept of MRgFUS-enabled brain tumor liquid biopsy and present the available preclinical evidence, ongoing clinical trials, limitations, and future directions of this application. From 2013 to 2017, the average annual age-adjusted incidence rate of all central nervous system (CNS) tumors in the United States was 23.79 per 100,000 people . Yet, dePoor prognoses for brain tumor patients can be partially attributed to the inadequate penetration of chemotherapeutic agents into the CNS due to the selective permeability of the BBB .Functionally, the BBB comprises endothelial cells, astrocyte end-feet, and pericytes, all of which are bound together via tight junctions that prevent the diffusion of most substances into the brain . TranspoPotential solutions to bypassing these limitations imposed by the BBB have included transiently disrupting the BBB or improving drug delivery into brain tumors using nanoparticle/microparticle drug carriers, peptide-based drug delivery, radiotherapy, or local delivery . AnotherBeyond limiting therapeutic access into the brain, the low permeability of the BBB additionally hinders the release of biomarkers of brain pathology into the peripheral circulation . Such moLiquid biopsy refers to detecting components in human fluids that are pathologically derived . Common analytes of liquid biopsies for cancer include circulating tumor cells (CTC), cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA) . ctDNA iAlthough ctDNA and cfDNA are the most common targets of liquid biopsies, other studied analytes include proteins, mRNA, miRNA, or extracellular microvesicles . Each ofSeveral publications have shown the possibility of detecting circulating brain tumor biomarkers –67. A suFocused ultrasound has been established as a BBB disruption technique to ablate tissue or enhance drug delivery. High-intensity focused ultrasound (HIFU) is used for brain tumor ablation by significantly increasing tissue temperatures using ultrasound energy , 83. Thitrans of gadolinium confirmed the BBB opening. Given the lack of pathology in their porcine model, their selected biomarkers were glial fibrillary acidic protein and myelin basic protein. After sonication, the researchers showed an increased concentration of those brain-specific biomarkers without major clinical or histological brain tissue damage. These summarized preclinical studies in animal models set the foundation for human clinical trials. There may be an optimal sonication pressure in patients that allows biomarker release with a low risk of hemorrhage like in the animal models. Likewise, higher sonication pressures may increase the risk of hemorrhage while the biomarker release plateaus.The combination of FUS and biomarkers was initially proposed by D’Souza et al. for human colon cancer lines in 2009 . Many suIn 2021, Meng and colleagues presented one of the first published evaluations of cfDNA detection enriched by MRgFUS in patients with pathologically confirmed GB . In theiThere are several completed and active clinical trials for the independent study of biomarkers in brain tumors or the safety of MRgFUS. For example, The Mayo Clinic is building a biorepository of CSF samples to analyze biomarkers (NCT04692324). Comparatively, the University of Maryland is assessing the safety and feasibility of ExAblate BBB disruption for treating high-grade gliomas in the context of standard chemotherapy (NCT03322813).Several other groups are following, and we are awaiting the results of vital clinical trials at the forefront of combining both liquid biopsy and FUS. For example, the BRAINFUL (BRAIN Tumor Focused Ultrasound-enabled Liquid Biopsy) Trial (NCT04940507) is a prospective, single-center, single-arm trial that hopes to enhance the detection of ctDNA by MRgFUS in glioma patients while understanding the changes in ctDNA over time. The BRAINFUL Trial, conducted by Dr. Lozano in Toronto, will analyze cfDNA in CSF and blood of patients that undergo tumor ablation with MRgFUS. Additionally, several grants have been recently awarded by the NIH to promote solid advances in this field. Amongst others, research teams at Washington University (1R01EB030102-01), Massachusetts General Hospital (5R01CA239078-02), and our team at Johns Hopkins, in collaboration with the University of Maryland (1R21NS113016-01), are working to revolutionize the detection and monitoring of brain tumors with the development of FUS-enabled liquid biopsies.The initiatives mentioned above will establish the foundation to translate FUS-enhanced liquid biopsy to the management of brain tumor patients. Those initiatives aim to overcome several current limitations. As described, available preclinical studies have used artificial biomarkers such as eGFP, which may not fully recapitulate the release of tumor associated biomarkers in the clinical setting. Additionally, terminal blood extractions shortly after sonication in animal experiments prevent the evaluation of FUS-induced biomarker release over time. Further studies are needed to assess the optimal sonication pressures and microbubble parameters for human brain tumor biomarker release while preserving safety. Evaluating biomarker release over time and with the influence of treatments such as radiation and chemotherapy is also paramount to understand the feasibility of FUS-enabled liquid biopsies. Tumor type, density, anatomical location, depth, skull physiology, surrounding vascular structures, and tumor-specific biomarkers may all require different parameters for optimal application of this evolving technique.The clinical application of liquid biopsies for cancer requires rigorous assay standardization, automatization, and technical and clinical validation to analyze a large volume of samples. At the same time, one of the main limitations for the brain tumor application of MRgFUS is the frailty of the brain parenchyma. Another consideration is that current MRgFUS systems are slow, expensive, and require customization. Given the need for such specialized technology, the economic burden of current detection strategies limits its uptake. With eagerness, the scientific community works to provide a faster, more reliable, and more affordable technology. Several academic and private institutions, such as the European Liquid Biopsy Society or the International Liquid Biopsy Standardization Alliance, coordinated by the Foundation for the NIH, are working to accelerate those much needed technological advances.Finally, ongoing clinical trials are often centered on advanced or recurrent brain tumors. Still, as knowledge and techniques progress, the goal is to detect early-stage cancers and improve the sensitivity to a point where early detection of recurrences precedes radiographic recurrence and promotes early intervention for improved survival in brain tumor patients.Many advances on liquid biopsies and MRgFUS for brain tumors have emerged over the past years. Discovering the possibilities of each independent method allowed us to explore the concept of FUS-enabled liquid biopsy. Understanding both techniques' biological and technological limitations gives us clear goals to overcome over the next decade. Some crucial aims are improving ctDNA detection methods and optimizing FUS settings to maximize biomarker release depending on the pathology and tumor location. Active research endeavors and clinical trials are underway. MRgFUS-enabled liquid biopsy is a revolutionary research field that promises a radical advance in diagnosing and monitoring brain tumors.


Trypanosoma cruzi. It is a very severe health issue in South America and Mexico infecting millions of people every year. Protozoan T. cruzi gets transmitted to human through Triatominae, a subfamily of the Reduviidae, and do not have any effective treatment or preventative available. The lack of economic gains from this tropical parasitic infection, has always been the reason behind its negligence by researchers and drug manufacturers for many decades. Hence there is an enormous requirement for more efficient and novel strategies to reduce the fatality associated with these diseases. Even, available diagnosis protocols are outdated and inefficient and there is an urgent need for rapid high throughput diagnostics as well as management protocol. The current advancement of nanotechnology in the field of healthcare has generated hope for better management of many tropical diseases including Chagas disease. Nanoparticulate systems for drug delivery like poloxamer coated nanosuspension of benzimidazole have shown promising results in reducing toxicity, elevating efficacy and bioavailability of the active compound against the pathogen, by prolonging release, thereby increasing the therapeutic index. Moreover, nanoparticle-based drug delivery has shown promising results in inducing the host’s immune response against the pathogen with very few side effects. Besides, advances in diagnostic assays, such as nanosensors, aided in the accurate detection of the parasite. In this review, we provide an insight into the life cycle stages of the pathogen in both vertebrate host and the insect vector, along with an overview of the current therapy for Chagas disease and its limitations; nano carrier-based delivery systems for antichagasic agents, we also address the advancement of nano vaccines and nano-diagnostic techniques, for treatment of Chagas disease, majorly focusing on the novel perspectives in combating the disease.Chagas disease, is a vector-mediated tropical disease whose causative agent is a parasitic protozoan named  T. cruzi  and amastigote (replicative form) in the vertebrate hosts. The trypomastigote form causes intercellular infections, upon entering the cells the trypomastigote transforms to amastigote form where it divides by binary fission. Amastigotes then leave the infected tissues and are transformed into metacyclic trypomastigotes in the bloodstream . In the 2+ ions , which is the most serious manifestation of this infection  loaded in multilamellar liposome vesicles (MLV) depicted much higher accumulation in the mice liver and lower blood BNZ concentrations compared to free BNZ treatment . Althougted mice . The use studies . Howeverin vitro assays using these nanoparticles loaded with nifurtimox showed very efficient anti-epimastigote activity, compared to free nifurtimox (40%. anti-epimastigote activity). Another study showed the use of sterols in polyethylene glycol-polylactide (PEG-PLA) nanospheres  was prepared by SEDDS comprising of isotropic mixtures of anhydrous oil and surfactant  is another compound of herbal origin, which have depicted very potent anti-trypanosomal activity in many assays, but owing to its low solubility and high lipophilicity, its use is restricted. Thus, LYC-loaded nanocapsules (NC) were prepared using poly-ε-caprolactone (PCL) and poly (lactic acid) polyethylene glycol (PLA-PEG) polymers, these nanocapsules depicted much higher drug efficacy in in vivo studies   , are lip (ISCOM) , namely  (ISCOM) . ISCOM e (ISCOM)  and werein vitro assays  no significant alterations were observed in the pathogen, moreover, their proliferation rate remained unaltered. Thus it can be said that although a lower concentration of CdTe QDs (2 μM) may be quite useful for bio-imaging, its use at a higher concentration is limited due to its toxicity (Quantum dots (QDs) nanoscale crystal structures that can transport electrons. When UV light illuminates these semiconducting nanoparticles, they can emit lights of various colors, this fluorescence properties make them an ideal nanomaterial for a large range of applications in biomedicine for drug delivery and diagnosis . But, tho assays , hence pstigotes , which rtoxicity .T. cruzi antigens from different chemical structures using polyunsaturated N-isopropyl acrylamide and trypan blue functionalized nanoparticles.Chagas disease can be diagnosed by two methods namely parasitological and serological , based oApart from this, another efficient nano-diagnostic biosensor systems are nanowires (NW), that are much more competent in comparison to their thin film analogs . The com® . Additionally, the liposomes carrying reconstituted membrane proteins of the parasite have proved to be a useful agent for immunization in many studies (A previous study has been reported whereby a single nanostructure was developed using (ISPA) immunostimulatory nanoparticles , that wa studies .T. cruzi has long been known as a potent antigen target (T. cruzi infection (®) in in vivo assays. The PLGA nanostructure aided in prolonging Tc24 antigen release. Moreover, this vaccine produced a rapid T-cell response. This report indicated the PLGA nanoparticles as a potent nanocarrier for hapten delivery that may elevate the efficacy as well as immunogenecity of a protein-based vaccine for Chagas disease (T. cruzi infection are their ability to attenuate the parasitic burden, tissue inflammation, as well as prevention of cardiac tissue damage, thereby causing an increase in host survival (Tc24 a protein found in the flagellar region of n target . Immuniznfection , 1995. Anfection . PLGA na disease . The dessurvival . Thus hesurvival .The nanotechnological strategies for drug and vaccine administration with the aid of nanocarriers embody a very potent strategy for Chagas disease treatment. This study made it very evident that a wide variety of nano-pharmaceutical drug delivery agents and diagnostic apparatus could open a new horizon of medical technology for the successful treatment of Chagas disease. However, the pre-clinical uses of these nano-medical technologies are encountered with some major challenges namely economic burden, especially for the developing countries, the elevated healthcare costs for this infectious disease produce an unfavorable cost-benefit ratio . Besidesin vivo studies, besides, these nanostructures have also increased the efficacy of the drugs. However, limited attempts have been made to explore the potential of these potent nanocarriers in pre-clinical studies. The use of nano-bio sensors, where nanodevice supplemented electrochemical immunoassay has also opened up new horizons for accurate and efficient detection of this infection at very early stages. Moreover, the use of nanomaterials to increase the immunogenicity of anti-chagasic vaccines can be very useful soon for the successful eradication of this infection. Although the use of nanotherapeutics against Chagas disease is in very nascent stages, appropriate study and application of these strategies against Chagas disease may play a pivotal role in the eradication of this infection.The danger of Chagas disease has long been unnoticed by both government and private pharmaceutical industries which hindered the pathway of new drug discovery and novel efficient treatment strategies against this infection. This zoonotic tropical infection has adversely affected both human and farm animal’s health in endemic regions and also poses a great danger for the near future if not controlled efficiently. The present review has shed light on the present treatments used against Chagas disease along with their limitations. It has majorly focused on the implication of nanotechnological interventions for enhancement of existing therapeutic options available for the treatment of Chagas disease. The nanocarriers encapsulated anti-trypanocidal drugs has shown enhancement of trypanocidal activity in both vitro and SC designed and wrote the complete manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gene delivery of antiviral therapeutics to anatomical sites where viruses accumulate and persist is a promising approach for the next generation of antiviral therapies. Recombinant adeno-associated viruses (AAV) are one of the leading vectors for gene therapy applications that deliver gene-editing enzymes, antibodies, and RNA interference molecules to eliminate viral reservoirs that fuel persistent infections. As long-lived viral DNA within specific cellular reservoirs is responsible for persistent hepatitis B virus, Herpes simplex virus, and human immunodeficiency virus infections, the discovery of AAV vectors with strong tropism for hepatocytes, sensory neurons and T cells, respectively, is of particular interest. Identification of natural isolates from various tissues in humans and non-human primates has generated an extensive catalog of AAV vectors with diverse tropisms and transduction efficiencies, which has been further expanded through molecular genetic approaches. The AAV capsid protein, which forms the virions' outer shell, is the primary determinant of tissue tropism, transduction efficiency, and immunogenicity. Thus, over the past few decades, extensive efforts to optimize AAV vectors for gene therapy applications have focused on capsid engineering with approaches such as directed evolution and rational design. These approaches are being used to identify variants with improved transduction efficiencies, alternate tropisms, reduced sequestration in non-target organs, and reduced immunogenicity, and have produced AAV capsids that are currently under evaluation in pre-clinical and clinical trials. This review will summarize the most recent strategies to identify AAV vectors with enhanced tropism and transduction in cell types that harbor viral reservoirs. Over the last few decades, the development of adeno-associated virus (AAV) as a vector for gene delivery has advanced significantly , 2. ReceA promising application of AAV-based therapeutics is the delivery of gene-editing enzymes to correct defective genes . In pre-AAV-mediated delivery of curative antiviral therapeutics to sites of persistent viral infection requires much of the same vector optimization as traditional gene therapy, including promoter/transgene optimization for expression at therapeutic levels, efficient transduction of target cells, and limiting the immune response to the vector and transgene . The simDependoparvovirus, family Parvoviridae, with a capsid containing 60 subunits of three different proteins  that have a common C-terminus and are produced via alternative translation of the AAV cap gene , and other species that exhibit a wide array of tissue and cell tropisms, determined mainly by the primary attachment receptor and co-receptor specificity of each serotype  cell cultures and included AAV2, the first to be vectorized. AAV2 vectors can transduce many tissue types, including liver, muscle, lung, retina, and brain , and moscap position 587/588 are now widely used to identify tissue-tropic mutant capsids. This approach has even been expanded to insert peptides at the same location in other serotypes such as AAV9 . SIn addition to the inherent potency and specificity of gene editing enzymes or RNAi activators for HBV, AAV vector optimization to maximize transduction and transgene expression in human hepatocytes is critical to gene therapy's effectiveness for viral infections. In recent years, significant advances in the development of liver-tropic AAV vectors have emerged from the gene therapy field that can also be harnessed to treat persistent hepatic infections . Due to Recent efforts to improve AAV vectors for liver-targeted applications have focused on three areas: (1) maximizing human hepatocyte tropism, (2) evasion of neutralizing antibodies, and (3) streamlining pre-clinical evaluations of liver transduction efficiency for novel AAV capsids. The first two have mostly focused on engineering the AAV capsid, which largely determines both tropism and antigenicity. The third is motivated by discrepancies in transduction levels observed in clinical trials compared to those obtained in pre-clinical studies in cell culture and animal models. Over the last few years, liver humanized mouse models have emerged as an important tool to study AAV transduction differences between serotypes at the pre-clinical stage. To effectively study antiviral therapies against HBV, liver humanized mice are crucial since HBV cannot replicate in most small mammals. In these xenograft models, primary human hepatocytes (PHHs) are transplanted into immunodeficient mice with genetic mutations that elicit murine liver injury. This deficit confers a growth advantage to PHH over murine hepatocytes, enabling high engraftment levels –126. StiAs previously discussed, AAV2 has successfully been used to treat blindness in clinical trials. In contrast, an AAV2 vector used for liver-targeted expression of FIX in early clinical trials for hemophilia B showed low efficacy, an interesting result given the robust transduction of human hepatocyte-derived cell lines by AAV2 vectors in vitro. A recent study suggests that tissue culture adaptations, possibly dating back to the early propagation of AAV2 in vitro, caused increased affinity for its primary cellular receptor heparan sulfate proteoglycan (HSPG). This resulted in enhanced transduction of primary hepatocytes and cell lines in vitro, while simultaneously reducing hepatocyte transduction in vivo . In the The generation of AAV capsid libraries followed by a selective screen has significantly increased the repertoire of AAV capsids with modified transduction properties. In 2014, Lisowski et al. used the humanized liver FRG mouse model to identify AAV capsid mutants with improved human hepatocyte transduction in vivo . This diSince AAV-LK03 was first characterized, several studies have reported conflicting results regarding the superiority of AAV-LK03 over other liver-tropic serotypes in mice with human livers and NHP , 129–131Although enhanced liver transduction efficiency has been seen with AAV-LK03 in some studies, this mutant is still moderately sensitive to pre-existing levels of neutralizing antibodies in humans . To direIn another directed evolution approach, intravenous immunoglobulin was passively transferred into liver humanized mice before administering the AAV capsid library to identify mutants that could evade AAV neutralizing antibodies and transduce human hepatocytes in vivo . After fIndividually, directed evolution and rational design have successfully created novel AAV vectors with enhanced liver-transduction capabilities and lower immunogenicity profiles. However, the combination of both methods has gained popularity in recent years , 134. ThIn summary, the identification of liver-tropic AAV vectors in pre-clinical settings using animal models and cell culture systems has been extensively optimized to select for variants that can both transduce hepatocytes at high levels and escape pre-existing immunity. As the liver is an important target for gene delivery in many gene therapy applications, humanized liver mouse models will continue to be used in the search for vectors that meet both criteria.Neurons of the peripheral nervous system (PNS) have long been identified as key target cells in the study of pain. They are also major targets for treatment of the incurable and persistent alphaherpesviruses HSV-1, HSV-2, and varicella zoster virus (VZV), which are highly prevalent amongst humans –138. AftThe capability to deliver anti-viral therapeutics to sensory neurons in vivo or influence them via overexpression of genes or inhibitors would be extremely valuable, and AAV vectors have been identified as useful tools to accomplish this. As discussed below, multiple studies have shown that localized delivery of AAV vectors to individual ganglia results in efficient gene delivery to sensory neurons. Unfortunately, the small size of sensory ganglia limits the effectiveness of this approach on a large scale as both somatic and autonomic ganglia of the PNS are found throughout the body in multiple distinct locations so that comprehensive gene delivery would require many localized administrations. For example, there are 62 dorsal root ganglia (DRG) in humans, two for each of the 31 cervical, thoracic, lumbar, and sacral nerve pairs that emerge from each side of vertebrate along the spinal column. As an alternative to localized delivery, efforts have been made to identify natural or engineered AAV capsid variants that can facilitate gene delivery to multiple sensory ganglia when delivered through a single vector administration route that results in more widespread delivery. Here we review studies that have investigated gene transfer to sensory ganglia following local or systemic AAV vector delivery.DRG houses neuronal cell bodies of the somatic nervous system that project from cervical, thoracic, lumbar, and sacral vertebrate and are involved in the relay of sensory information from peripheral sites to the central nervous system (CNS). DRG have been identified as a primary target for pain, spinal cord injury, and neurodegenerative disease studies. These clusters of neurons are also major 'reservoirs' for viral DNA during latent VZV and genital HSV infections –141, as Localized AAV delivery routes such as direct lumbar or sciatic nerve injections can facilitate efficient gene transfer to individual or multiple closely situated DRG. Following localized injection directly into the DRG or the sciatic nerve, AAV vectors including serotypes 1, 2, 5, 6, 7, 8, 9, rh10, and an engineered AAV2-retro vector can all efficiently transduce neurons of the DRG in mouse, rat, and pig animal models –148. In Indirect uptake via localized AAV delivery to afferent nerve terminals that project from peripheral sites has shown promise as an approach to transduce multiple ganglia. In one such study, AAV8-GFP vectors were injected into the footpad of mice, resulting in the transduction of > 90% of sensory neurons in multiple DRG that innervate the footpad . FurtherDelivery of therapeutic transgenes to multiple DRG via the cerebrospinal fluid (CSF) or blood offers a more straightforward route to widespread gene delivery in multiple DRG, and several groups have investigated this. When delivered via intrathecal (IT) injection, AAV vectors can efficiently transduce sensory neurons in multiple DRG , 151–156Intravenous delivery of AAV vectors should, in theory, enable gene delivery to any cell within close proximity of the vasculature. However, unlike IT administration, IV delivery results in the circulation of AAV vectors through organs that are not part of the nervous system, such as the liver and spleen, where vector is often sequestered at high levels before it has a chance to see the target organ. Subsequently, sensory DRG likely see a lower relative effective AAV dose via IV delivery than IT, and mostly, AAV vectors do not appear to transduce sensory ganglia as efficiently through this administration route. Unfortunately, non-specific sequestration is inherent to most naturally occurring AAV capsids. However, one approach to prevent this has been to screen AAV capsid libraries for engineered variants that can transduce sensory ganglia when delivered IV to bypass non-specific sequestration. In vivo library selection has been used to identify AAV capsids with enhanced tropism for neurons of the CNS and PNS with great success and has yielded at least one novel capsid (AAV.PHP.S) that can efficiently transduce up to 82% of DRG neurons in mice after IV delivery , 61, 157In mammals, two trigeminal ganglia (TG) sit below the brain, and house cell bodies of tactile, proprioceptive, and nociceptive afferent somatic sensory neurons that innervate the face. These TG are responsible for relaying craniofacial sensory and motor nervous stimuli from the left or right side of the face into the CNS through a large sensory root and small motor root that enters the brainstem. Like the DRG, the TG is a target for studies of pain, but it is also a reservoir for viral DNA during latent VZV and craniofacial HSV infection –141, andDirect injection of AAV delivery into the TG is extremely challenging due to its location immediately below the brain. Therefore, attempts to transduce sensory neurons of the TG using AAV vectors have mainly focused on indirect vector transductions methods involving delivery via projecting efferent neurons of the face or IV administration. Transduction of the TG has been attempted via delivery of AAV to the eye since efferent neurons from the TG project into the conjunctiva of the eye, but species-specific differences have been seen in delivery efficiency. In mice, the delivery of AAV to the eye has proved inefficient for TG delivery with minimal transduction of sensory neurons seen . ConversThe autonomic nervous system (ANS) is a branch of the PNS that unconsciously controls many bodily functions and can be divided into sympathetic 'fight-or-flight' neurons, parasympathetic 'rest-and-digest' neurons, and enteric neurons that control motor functions of the GI tract. The potential to transduce autonomic neurons would be useful for studying many aspects of neurological function across multiple body systems, and AAV vectors have been investigated to this end. Like somatic neurons, autonomic neurons of the sympathetic, parasympathetic, and enteric nervous systems can also harbor latent alphaherpesvirus DNA within neuronal cell bodies of distinct ganglia –162, so While more limited in number than studies in the somatic nervous system, some studies have shown that ANS neurons can be efficiently targeted with AAV vectors. In mice, AAV8 and AAV9 vectors can efficiently transduce up to 57% of myenteric and submucosal neurons of the GI tract when delivered systemically via IV injection , 164. InDespite the promise shown by AAV vectors for gene delivery to sensory neurons in vivo, several recent studies in large animals suggest that IV or IT delivery of high doses of AAV vectors may lead to asymptomatic injury to sensory neurons in vivo. Mononuclear cell infiltration and minimal to moderate asymptomatic degeneration of DRG neurons and associated axons has been seen in NHP, and more severe sensory neuronal lesions were seen in pig DRG –169. WhiCD4+ T lymphocytes are hematopoietic cells that play a major role in controlling infectious pathogens during the initiation of humoral and cellular immune responses by the host. Due to the many different and complex roles they play in host immune responses, CD4+ T cells have been identified as important targets in the study of autoimmune diseases, such as the severe combined immunodeficiencies (SCID), and for the development of cell-based immune-mediated therapeutics, such as chimeric antigen receptor (CAR) T cells. CD4+ T cells have also been identified as important targets for gene therapies targeting HIV, which selectively infects CD4+ T cells and CD4+ myeloid cells after infection and establishes lifelong infections via integration of its genome into the host chromosome. Unlike hepatocytes or sensory neurons, CD4+ T cells are ubiquitously found throughout the body in blood and other lymphoid and non-lymphoid tissues, which makes their transduction in vivo for the treatment of HIV highly challenging. This is of high importance for developing curative gene therapies against HIV that require widespread CD4+ T cell gene transfer to every infected cell throughout the body. Fortunately, despite the observed toxicity in the DRG, AAV vectors have generally shown a good safety profile when delivered systemically at high doses in several animal models, so they are being investigated as tools to deliver different types of therapeutics to CD4+ T cells both in vivo and ex vivo.CD4+ T cells can be isolated from whole blood and transduced with gene therapeutics ex vivo before transplantation, or they can be directly targeted in vivo in blood and tissues when vectors are delivered systemically. For some therapeutic purposes, CD4+ T cells can also be targeted indirectly via transduction of hematopoietic progenitor cells that can differentiate into the lymphocyte lineage and become CD4+ T cells. AAV vectors have shown promise for all these approaches to T cell transduction, and AAV capsids showing promise for CD4+ T cell delivery have included naturally occurring capsids and capsids identified via directed evolution or rational design.Initial attempts to determine whether naturally occurring AAV vectors could transduce hematopoietic cells identified AAV6 as a serotype that could efficiently transduce mouse myeloid cells . SubsequApproaches to identify capsids that transduce T cells using directed evolution or rational design have been few but show promise. In one study, AAV2 error-prone PCR, AAV2 loop insertion, and AAV shuffle libraries were pooled and screened for variants with improved transduction of the H9 T cell line . Capsid SaCas9 gene in combination with sgRNAs that target the HIV or SIV long terminal repeat (LTR), and/or HIV gag can be used to excise portions of the HIV provirus in vivo when delivered systemically. HIV provirus excision was demonstrated in these studies in whole blood, CD4+ T cells, lymphoid, and non-lymphoid tissues throughout the body following a single AAV vector infusion [While efforts continue to identify better capsids that efficiently target CD4+ T cells, several recent animal studies have shown that systemic delivery of AAV-based therapeutics may offer a realistic pathway to effective anti-HIV therapies. Studies in HIV transgenic mice, humanized BLT mice infected with HIV, and SIV infected macaques have shown that AAV9 or AAV-DJ vectors expressing the infusion , 183, aninfusion . These sAcross the global human population, multiple persistent viral infections affect billions of people. While some of these infections are not associated with disease, or only cause disease in immunocompromised individuals, the proportion of viral infections that causes disease is of significant medical importance . For insAAV vectors are a promising tool to achieve this because of their lack of pathogenicity, long-term transduction capacity, and relatively low immunogenicity, given their widespread natural prevalence in humans. As discussed here, the preclinical development of AAV vectors via capsid engineering in recent years has resulted in vectors with enhanced tissue tropism and potent transduction efficiencies in the liver, the peripheral nervous system, and CD4+ T cells, demonstrating the potential of AAV vectors to access anatomical sites harboring viral reservoirs. Nonetheless, significant challenges remain and have been best illustrated by studies in large animal models and clinical trial outcomes. A substantial obstacle for the implementation of AAVs as gene therapy vectors is the immunogenicity resulting from pre-existing anti-AAV antibodies, which many groups have undertaken preclinically by in vivo selection of AAV capsid libraries using directed evolution, rational design, or a combination of both. Other approaches to circumvent pre-existing immunity have explored AAV-specific plasmapheresis for the selective removal of circulating antibodies that can neutralize AAV vectors and the administration of immunosuppressive drugs before AAV administration. However, the effectiveness of these approaches has yet to be evaluated in humans –188.The fast-paced preclinical development of novel AAV capsids for specific applications has significantly expanded the AAV catalog and holds promise for identifying vectors with desired properties for different applications. However, the extensive AAV ‘toolkit’ will require head-to-head comparisons of immunogenicity, biodistribution, and cell/tissue/organ transduction efficiencies to narrow down the best vectors to be evaluated clinically.


Cardiovascular magnetic resonance is increasingly used in clinical practice, as it has emerged over the years as an invaluable imaging technique for diagnosis and prognosis, with clear-cut applications in managing patients with both ischemic and non-ischemic heart disease. In this review, we focus on the evidence and clinical application of stress CMR in coronary artery disease from diagnosis to prognosis.  Cardiovascular magnetic resonance (CMR) is a non-ionizing, multi-planar imaging modality that is increasingly used in clinical practice, as it is the gold standard for biventricular volumes and function assessment and is capable of accurate non-invasive myocardial tissue characterisation. Through excitation, inversion, and saturation pulses, CMR exploits the intrinsic properties of normal and abnormal myocardial tissue components  and images are therefore weighted to express different pathologic processes: for example, T2-weighted images are used to detect myocardial oedema, while T1-weighted images are used to detect fibrosis and fat infiltration [CMR uses gadolinium-based contrast agents (GBCAs) that are administered intravenously and have an extra-cellular and extra-vascular distribution so that they accumulate in expanded myocardial interstitial space. GBCAs reduce T1 times and can be detected on T1-weighted images acquired 10–20 min after administration as areas of myocardial signal enhancement . Ventricular distribution pattern of LGE allows an accurate identification of myocardial damage suggesting the underlying etiopathogenesis: ischemic LGE patterns follow coronary artery distribution and the ischemic wave-front, thus initially involving the sub-endocardium and subsequently extending through the myocardial wall to become transmural in cases of prolonged myocardial ischemia; non-ischemic patterns do not follow coronary artery distribution and generally spare the sub-endocardium, having a sub-epicardial and/or mid-wall distribution ,4,5. Patients referred to CMR have to be carefully screened for potential contraindications to a CMR scan. CMR is contra-indicated in patients wearing metallic devices, such as vascular  clips, ocular metallic bodies, and implanted pumps for medication infusion. Coronary stents can be scanned immediately after implantation up to 3 T, as the vessel wall provides stent stability after deployment; however, metallic artefacts do not allow the evaluation of the stent itself . Most ofSystemic nephrogenic sclerosis (NSF) is the most dangerous complication potentially related to the administration of GBCAs and has been mostly reported in patients with advanced renal disease. A recent meta-analysis on 4931 patients with stage 4 and 5 chronic kidney disease (CKD) who received American College of Radiology classification group II GBCAs , which are known to be at very low risk of NSF, found a pooled incidence of NSF of 0, with an upper bound of risk of 0.12% to 1.59% using different group II GBCAs [Although group II GBCAs administration in CKD patients appears safer than in the past, the advent of parametric mapping, which provides a pixel-wide, colour-coded map reflecting intrinsic myocardial tissue properties, allows safe and diagnostic CMR scanning without the need of contrast agents . Pharmacological stress CMR can be performed using both vasodilator   and inotInducible myocardial perfusion defect is visually defined on three short axis images  as an area of subendocardial/transmural hypo-perfusion occurring first when the contrast reaches the left ventricular myocardium (first pass) and persisting for >4 RR intervals, more than 1 pixel wide, and conforming to the distribution territory of one or more coronary arteries  Figure . True, iAlthough visual assessment of myocardial ischemia prevails in clinical practice, semi-quantitative and quantitative assessment of myocardial perfusion is also possible, is based on the analysis of signal intensity curves acquired during the first wash-in of contrast agent ,13,14 anVery recent technical advances using automatically inline artificial intelligence for quantification of stress CMR perfusion mapping allow a robust quantification of myocardial blood flow (MBF) and myocardial perfusion reserve (MPR), which are strong, independent predictors of adverse cardiovascular outcomes in patients with known or suspected coronary artery disease .Stress CMR also allows the identification of peri-infarct ischemia, which is generally defined as a reversible myocardial perfusion defect adjacent to and larger than infarct-related scar; although its mechanism is still debated, peri-infarct ischemia has been related to increased apoptosis; it is thought to be involved in pathological ventricular remodelling and has been shown to be associated to ventricular arrhythmias . Vasodilator agents induce myocardial ischemia through the coronary steal phenomenon: blood flow is increased to areas supplied by patent coronaries while being simultaneously reduced to areas supplied by stenotic coronaries. Differences among vasodilator agents are reported in Termination criteria of vasodilator stress testing include persistent or symptomatic AVB, a significant drop in systolic pressure (>20 mmHg), persistent or symptomatic hypotension, and severe respiratory difficulty. Vasodilator stress perfusion CMR has been shown to be safe in patients with HF and reduced left ventricular ejection fraction (LVEF < 40%), with no adverse events reported in more than 1000 patients . SimilarDobutamine stress CMR increases myocardial contractility through beta-1 stimulation, and can be used both to assess viability (at low doses) and ischemia (at high doses). Dobutamine stress CMR should not be performed in patients with serious hypertension >220/120 mmHg), in patients with unstable angina, acute myocardial infarction, severe aortic stenosis, obstructive hypertrophic cardiomyopathy and narrow angle glaucoma, and complex cardiac arrhythmias, including uncontrolled atrial fibrillation, and in patients with myocarditis, endocarditis, or pericarditis. Termination criteria include achievement of target heart rate, a significant drop in systolic pressure (>20 mmHg), persistent symptoms, symptomatic tachycardia, and serious hypertension [/120 mmHgAs stressor agents carry the risk of potential life-threatening arrhythmias, physicians running a stress CMR service have to be trained in advanced cardiovascular life support and have to be familiar with the crash trolley that has to be readily available . ECG traExercise CMR has developed over the years, and it is nowadays used more frequently  than exeStress perfusion CMR has been established as a valuable diagnostic and prognostic tool and is recommended by international guidelines in the assessment of patients with coronary artery disease (CAD) . The 2019 European Society of Cardiology (ESC) guidelines on chronic coronary syndromes (CCS) recommend the use of stress CMR as the initial test to diagnose CAD in symptomatic patients in whom obstructive CAD cannot be excluded by clinical assessment alone  . These gBoth 2018 ESC guidelines on myocardial revascularization  and 2016The 2015 ESC guidelines on non-ST elevation myocardial infarction recommend the use of non-invasive imaging stress tests in patients with no recurrence of chest pain, normal ECG, and normal levels of high-sensitive troponin but suspected acute coronary syndrome before deciding on performing an invasive test  ; patientThe ISCHEMIA trial, a large trial on 5179 patients with moderate or severe myocardial ischemia on stress test, has shown the pivotal role of optimal medical therapy and the lack of benefit of early invasive strategies at a median follow-up of 3.2 years; however, a subgroup analysis excluding procedural infarction has suggested better outcome on longer follow-up (up to five years) in patients undergoing coronary revascularization than those receiving optimal medical therapy. Of note, as the choice of the specific test for ischemia detection was based on physician preference or local availability, only 5% of enrolled patients had ischemia detected by stress CMR as compared to nearly 50% detected by SPECT; therefore, the study is likely not powered to assess the prognostic role of stress CMR . NeverthEvidence of the diagnostic role of stress perfusion CMR has significantly increased over the last two decades. In the late 2000s the multi-centre, multi-vendor MR-IMPACT trial , by compThe CE-MARC trial recruited 752 patients with suspected angina and at least one cardiovascular risk factor undergoing adenosine stress perfusion CMR, SPECT, and coronary angiography as reference standard: CMR showed high diagnostic accuracy for the detection of CAD and proved to be superior to SPECT both in single and multi-vessel disease . A sub-aMore recently, the MR-INFORM trial showed that stress perfusion CMR is associated with a lower incidence of coronary revascularization than invasively assessed fractional flow reserve (FFR)  and that CMR is non-inferior to FFR with regards to the incidence of major adverse cardiac events [CT), a novel, non-invasive functional test, has recently prompted the comparison with stress perfusion CMR for prediction of coronary revascularization in patients with stable angina and one or more coronary stenosis (≥50%) on CT. A recent study from Rønnow Sand NP et al. showed that, although FFRCT had higher sensitivity than perfusion CMR  to identify patients needing coronary revascularization, stress perfusion CMR had higher specificity (88% vs. 42% by FFRCT) and positive predictive value (67% vs. 47%) [The advent of computed tomography-derived FFR (FFRvs. 47%) . Semiquavs. 47%)  both at CT, and no imaging [Additionally, stress perfusion CMR has been shown to be a cost-effective test in different international studies. Stress perfusion CMR has been compared to immediate coronary angiography with selected FFR, SPECT, coronary CT with FFR imaging  in a Uni imaging .Besides the described role of stress CMR over other imaging modalities in the detection of myocardial ischemia, it has to be noted that CMR carries other clinically-relevant advantages: it does not use ionizing radiations , it has higher contrast-to-noise ratio than CT when assessing myocardial perfusion, and has good temporal resolution but lower spatial resolution as compared to CT ,40.Given the high diagnostic yield of stress CMR and considering that it is a very comprehensive examination able to provide information on ventricular function, stress myocardial perfusion, and viability within a single study, much effort has recently been made to provide reliable evidence on its prognostic value ,41. The 2), in patients with atrial fibrillation and suspected or stable coronary artery disease, as well as in elderly patients (>75 years) with suspected coronary artery disease [Finally, many studies tried to estimate the prognostic role of stress CMR in specific populations. In a sub-analysis of the SPINS study, focused on patients with reduced LVEF <50%), the authors showed that the presence of ischemia, LGE, or both was associated with higher event rates and that both of them were independent predictors of primary  and secondary outcomes  . These f disease ,49,50 an%, the auSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic of respiratory disease since late 2019 (referred to as COVID-19 pandemic), determining millions of deaths to date . ClinicaStress CMR is a safe and feasible test for assessing patients with known or suspected coronary artery disease, and this holds true also in specific categories of subjects, such as women, obese patients and patients with severe left ventricular dysfunction, atrial fibrillation, left bundle branch block. Currently, vast amounts of clinical data support the evidence on diagnostic and prognostic role as well as the cost-effectiveness of CMR-based clinical strategies. Unfortunately, availability of dedicated CMR scanners and of experienced operators may still be limited on a local base. On the other hand, increasing recognition of the key role of CMR in the assessment of many non-ischemic cardiac conditions, in many cases, is acting as a practical stimulus for clinical cardiologists to consider stress CMR as a valuable alternative to other tests for non-invasive ischemia detection.