Clonorchis sinensis, and is often associated with a malignant form of bile duct cancer (cholangiocarcinoma). Although some aspects of the epidemiology of clonorchiasis are understood, little is known about the genetics of C. sinensis populations. Here, we conducted a comprehensive genetic exploration of C. sinensis from endemic geographic regions using complete mitochondrial protein gene sets. Genomic DNA samples from C. sinensis individuals (n = 183) collected from cats and dogs in China  as well as from rats infected with metacercariae from cyprinid fish from the Russian Far East (Primorsky Krai region) were deep sequenced using the BGISEQ-500 platform. Informatic analyses of mitochondrial protein gene data sets revealed marked genetic variation within C. sinensis; significant variation was identified within and among individual worms from distinct geographical locations. No clear affiliation with a particular location or host species was evident, suggesting a high rate of dispersal of the parasite across endemic regions. The present work provides a foundation for future biological, epidemiological and ecological studies using mitochondrial protein gene data sets, which could aid in elucidating associations between particular C. sinensis genotypes/haplotypes and the pathogenesis or severity of clonorchiasis and its complications (including cholangiocarcinoma) in humans.Clonorchiasis is a neglected tropical disease caused by the Chinese liver fluke,  Clonorchis sinensis, which can induce malignant bile duct cancer (cholangiocarcinoma). Little precise information is available on the biology, epidemiology and population genetics of C. sinensis. For this reason, we explored here the genetic composition of C. sinensis populations in distinct endemic areas in China and Russia. Using a deep sequencing-informatic approach, we revealed marked mitochondrial genetic variation within and between individuals and populations of C. sinensis, with no particular affiliation with geographic or host origin. These molecular findings and the methodology established should underpin future genetic studies of C. sinensis causing human clonorchiasis and associated complications (cancer) as well as transmission patterns in endemic regions.Clonorchiasis is an important neglected tropical disease caused by the Chinese liver fluke,  Clonorchis sinensis . Chronic infection with this fluke can induce cholangiocarcinoma in humans–a malignant and fatal form of bile duct cancer )ternally –70. Althternally , 71, 72 agonimus , 74, and elegans , 75, whi elegans . Althougificity; , 67). Fuificity;  to eluciquencing  to eluciC. sinensis, which might reflect an ‘adaptive capacity’ in this species, particularly for factors linked to the establishment of infection. It is generally accepted that the adaptive capacity of a species is reflected in the levels of genetic diversity within populations, as a high level of variation provides a mechanism to respond to different selection pressures in the environment; such variation might be reflected in phenotypic variation. Phenotypic traits of particular interest in parasites, relevant to understanding parasite biology and potentially parasitic diseases, include differences in morphology, immune modulation or evasion, host preference or affiliation, sensitivity to drugs, virulence or pathogenicity, and/or adaptation to geographical regions with particular climatic conditions or host spectra. Investigating the impact that genetic variation within C. sinensis populations has on such traits relies on comprehensive genetic data and relevant biological, ecological, epidemiological or clinical information available upon sample collection. Our current data sets enabled us to investigate whether a genetic composition of adult C. sinensis populations correlated with geographical and/or known naturally infected definitive host species (cat or dog), as the worm isolates were obtained from disparate northern and southern zones in which C. sinensis is endemic [In the present study, numerous fixed nucleotide variations were detected between some individuals of  endemic .C. sinensis is similar across the distribution range of this species, which would suggest high levels of ongoing gene-flow between and among populations, probably due to the migration of humans and/or domestic or wild canids or felids, and possibly the transport of intermediate hosts or infected food products. This is concerning in the sense that, if new, evolutionarily advantaged phenotypes emerged , there would be a marked potential for such variants to be spread across the distribution range of C. sinensis; it might also facilitate and enhance parasite transmission or dissemination in endemic or newly established foci of infection. It is also interesting to note that the apparent endemic hot-spots for C. sinensis in the ‘north’ and ‘south’ might differ in their epidemiology  linked to climatic conditions , known to have a significant impact on the success of trematode transmission, presence, prevalence and biology of intermediate host species, such as higher temperatures enhancing snail growth, activity and survival [C. sinensis in these regions and what impact high human migration rates might have on these possible local adaptations  remain C. sinensis from cats and dogs appears to be indicative of a plasticity in adaptation to definitive host species. Not only were the samples obtained from the two host species genetically similar, remarkably, several of them were genetically identical in their mitochondrial protein-encoding sequences , in addition to cats and dogs [C. sinensis in humans to establish whether there is a genetic differentiation between isolates from people and other host species, and whether particular genotypes/haplotypes have an association with the pathogenesis, the severity of disease and complications, such as cancer, in humans. Such an endeavour would be reliant on obtaining worms during surgery, which might be feasible in endemic regions, such as southern China , and the Russian Far East. Alternatively, eggs from human faecal samples could be collected during epidemiological studies and subjected to genetic analysis–preferably at an individual egg level. Given the knowledge gaps regarding the genetic composition of C. sinensis in human populations, finding unique variants of C. sinensis could have significant implications for transmission and control . It seeC. sinensis in the present study and the establishment of an integrated deep-sequencing-informatic-phylogenetic approach provide a solid foundation to tackle these research questions or areas. Clear proof-of-principle of this technological approach also opens the door to large-scale and comparative population genetic and molecular epidemiological analyses using whole mitochondrial and nuclear genomic data sets.Clearly, the finding of marked genetic variability within S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 FigClonorchis sinensis individuals studied here and four published mitochondrial genomes  employing Bayesian (BI) and maximum likehood (ML) methods. Specimen numbers are indicated , haplotJ381664; ), haplotJ381664; ), and haJ381664; ). Cluste(TIF)Click here for additional data file.


Autoimmune uveitis is a major cause of blindness in humans. Activation of retina-specific autoreactive T cells by commensal microbiota has been shown to trigger uveitis in mice. Although a culprit microbe and/or its immunogenic antigen remains to be identified, studies from inducible and spontaneous mouse models suggest the potential of microbiota-modulating therapies for treating ocular autoimmune disease. In this review, we summarize recent findings on the contribution of microbiota to T cell-driven, tissue-specific autoimmunity, with an emphasis on autoimmune uveitis, and analyze microbiota-altering interventions, including antibiotics, probiotics, and microbiota-derived metabolites , which have been shown to be effective in other autoimmune diseases. We also discuss the need to explore more translational animal models as well as to integrate various datasets  to gain a better understanding of how microbiota can directly or indirectly modulate the immune system and contribute to the onset of disease. It is hoped that deeper understanding of these interactions may lead to more effective treatment interventions. Development of autoimmune diseases is influenced by both genetic and environmental factors . In geneCommensal microbiota colonize the barrier surfaces of our body that come into contact with the environment . The mosThe eye and brain are immunologically privileged sites. The target antigens are sequestered behind the blood-organ barrier and are not available outside the tissue to prime self-reactive T cells that have escaped from thymic negative selection and are present in circulation . The funT cells play a major role in the pathogenesis of autoimmunity. Th1 and Th17 cells have been intensively studied and shown to have effector and pathogenic roles , whereasThe need for mechanistic understanding of microbiota-host interactions stimulated studies in which the identification of culprit microbes has yielded associations between commensals and effects on host immunity . HoweverInvolvement of the microbiome in the development of tissue-specific autoimmune diseases has sparked interest into understanding the crosstalk between microbiota and host immunity, and how this influences overall health . During Uveitis is a general term referring to ocular inflammatory disorders of the uveal tract and one of the leading causes of the vision loss . AutoimmAmong the experimental mouse strains tested, B10.RIII is the most highly susceptible to experimental autoimmune uveitis (EAU) induced by active immunization with the retinal antigen, interphotoreceptor retinoid binding protein (IRBP), without need for additional stimulation with pertussis toxin . Our labMicrobiota may also modulate uveitis in ways other than providing a source of surrogate antigen. In immunization-induced EAU, where disease is triggered by antigen administered peripherally, oral antibiotic treatment (ABX) has the potential to inhibit disease. However, different groups reported conflicting results: long-term ABX in wild-type B10.RIII mice did not affect EAU development following IRBP immunization , but shoAkkermansia muciniphila and Acinetobacter calcoaceticus) that were enriched in MS patients induced proinflammatory responses in human peripheral blood mononuclear cells and in mono-colonized mice , a model for multiple sclerosis (MS), a disease affecting the brain and spinal cord which are also considered immune privileged tissues . The eyezed mice .Bacteroides thetaiotaomicron, a commensal shown to have been enriched in myocarditis patients, expressed a β-galactosidase peptide that mimics the host protein MYH6 and activates antigen-specific T cells that enhance disease , a target antigen expressed in pancreatic beta cells. NOD mice expressing a TCR specific to IGRP in their CD8+ T cells developed highly accelerated diabetes when associated with gut microbiota enriched with Fusobacteria  in non-obese diabetic (NOD) mice. A transgenic mouse model that expresses a TCR specific for myosin heavy chain 6 (MYH6) was reported to develop spontaneous cardiomyopathy.  disease . In NOD bacteria . Neverthbacteria . Anotherbacteria .Reports showing associations between specific gut commensals and human autoimmune diseases continue to multiply. A detailed enumeration of the particular microbes that are associated with diverse diseases is beyond the scope of this article, and the subject has recently been reviewed . It shouIt is very difficult to rigorously establish cause-and-effect relationships in human studies. Published studies on the role of microbiota in human uveitis fail to establish causality. Nevertheless, many reports are suggestive, and hypotheses can be supported by well controlled experiments in animal models.Early studies linking gut microbiota to autoimmune diseases were performed by direct perturbation of the microbiota in different experimental models. If there is a positive correlation between the microbiota and progression of disease, ABX or GF rearing is expected to abrogate disease progression . HoweverABX can also have unintended effects on host immune responses. As discussed above, duration of ABX can result in different disease outcomes in the EAU model. A separate study investigating the effects of ABX on host immune status in mice showed that body weight and spleen size were decreased, while cecum  was enlarged within 1 week of ABX. However, after the 1st week, continued ABX did not seem to worsen gut inflammation, and colon enterocyte transcriptome was restored to a state closer to non-treated controls . Thus, iAlthough, unlike GF mice, ABX animals have the advantage of a fully developed immune system, ABX does not eliminate fungal or viral species. Therefore, ABX models cannot rule out the immune interactions between the host and commensal fungi/viruses, confounding correlation studies.In hopes of extrapolating results from animal models to human subjects, a “humanized” approach is often applied, in which human microbiota is established in GF or ABX animals . A typicBesides metabolic syndrome to which diet is often the key contributing factor, gut microbiome has been shown to influence a range of autoimmune diseases in sites distant from the gut, such as rheumatoid arthritis , autoimmPrevotella histicola (Ongoing or future efforts of HMA studies are to 1) mono-colonize GF animals with candidate microbes identified from health-disease comparisons to explore the potential contribution of individual microbes to the development of pathologies; and 2) evaluate whether certain microbes could serve as novel therapeutics to treat diseases, e.g., suppression of arthritis or EAE in humanized mouse models when treated with isolated human gut commensal isticola .Though the HMA model remains a powerful tool to study disease-microbiome associations, it may have limitations in translatability. Gnotobiotic mice harboring microbiota from heterologous species  revealed an immature immune system, which failed to serve as a counterpart to human immunity . DiffereTo take this concept even further, some studies indicate that laboratory SPF mouse flora is also not equivalent to the natural co-evolved mouse flora. SPF mice typically contain fewer memory cells than their wild-caught counterparts and in that regard their adult immune systems are more reminiscent of neonatal humans . This maNot surprisingly, the concept of an antigen-experienced, “dirty” mouse model, long known to be more robust immunologically than SPF mice, has been receiving growing attention as a better mimic of the diverse history of infections typical of humans . RecentlSince microbiome-disease association studies typically involved antibiotics treatment, and because of the simplicity and ease of bacteria-specific 16S marker gene survey to characterize the microbiome, attention has mostly been focused on the bacterial components within the microbial consortia. However, host-associated microbiota encompass all domains of life including bacteria, archaea, fungi, viruses and protozoa. In particular, the involvement of fungal and viral species in autoimmunity has been increasingly appreciated, strengthening the long-standing observation that fungi and viruses have the potential to trigger autoimmune diseases .Candida albicans, a well-characterized fungal pathobiont in the human gut , prebiotics  or in more extreme cases fecal microbial transplant (FMT), which has become popularized by its high degree of success in curing ysbiosis . The useysbiosis . Yet, hoysbiosis . There iysbiosis . Althougis cases .Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus reuteri, Bifidobacterium bifidum, and Streptococcus thermophilus) was examined for ability to treat immune-mediated eye disease models. It showed therapeutic effects on autoimmune uveitis and dry eye, but had no effect on corneal graft survival. To facilitate engraftment of the probiotics, investigators pretreated mice with antibiotics for 5 days , serve as fingerprints of microbial function, and can act systemically on the host via absorption into the circulation and tissues . SCFAs cBesides probiotics and microbial metabolites, phage therapy has also come under the spotlight in an effort to combat the rising antibiotic resistance of bacteria . In addiThe understanding of “-omics” aims to make sense of the collective characterization and quantification of biological components that translate into the structure, function, and dynamics of an organism or organisms . Large din silico approaches, if the T and/orB cell autoepitopes are well characterized  and T/B cell receptor or MHC binding  progression and treatment response . Plasma Metabolomic studies can also be applied to the interrogation of intestinal microbiota (metametabolomics) . MetametThe entire suite of regulatory components in a cell is named the regulome, and its study has focused on processes by which a set of genes are regulated during development or under different physiological and pathological states . AlthougThe limitations of the “-omics” approaches, with the possible exception of metagenomics, are the high cost per sample and the requirement of high-level bioinformatics expertise and computational power to be able to process “big data”. Concerning uveitis, so far metagenomic and metabolomic profiles on patients have been studied , but we With the popularization of multi-omics technology, increasing numbers of studies incorporated metagenomic, metatranscriptomic, metaproteomic or metametabolomic analyses, the integration of which has led to unprecedented insights into mechanisms of disease-microbiome association . In a reHost-associated microbiota represent a dynamic ecosystem of commensal/pathogenic microbes, which are influenced by the environment and in turn affect our health. Experiments in laboratory-reared “clean” mice may not faithfully represent the human condition, as they cannot recapitulate the heterogeneity in genetic make-up and microbiota status in humans. Animal models such as “rewilded” and “wildling” systems, or possibly models with a “humanized” immune system, may help bridge this gap in some cases. In addition, it may be worth performing a thorough analysis of human patient samples to identify biomarkers of interest, prior to interrogating related pathways in animal models to determine disease mechanism.Traditional approaches, including 16S metagenomic sequencing and immunological characterization, can be further enhanced by incorporating metabolic data from plasma or serum. Similar to how databases are being standardized, there is an urgent need to develop methods to analyze and integrate data from various sources in a systematic manner. Potentially, open-source analytical pipelines and containerized bioinformatics serve as a great opportunity to bridge the gap between big data and meaningful output of results. If implemented effectively, a systems biology approach and the integration of big data may yield new and exciting hypotheses to be tested.RS and AZ made the outline and wrote the manuscript. RH and RRC supervised, edited, and 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.


Neuromuscular blocking agents (NMBAs) can be an effective modality to address challenges that arise daily in the intensive care unit (ICU). These medications are often used to optimize mechanical ventilation, facilitate endotracheal intubation, stop overt shivering during therapeutic hypothermia following cardiac arrest, and may have a role in the management of life-threatening conditions such as elevated intracranial pressure and status asthmaticus (when deep sedation fails or is not tolerated). However, current NMBA use has decreased during the last decade due to concerns of potential adverse effects such as venous thrombosis, patient awareness during paralysis, development of critical illness myopathy, autonomic interactions, and even residual paralysis following cessation of NMBA use.It is therefore essential for clinicians to be familiar with evidence-based practices regarding appropriate NMBA use in order to select appropriate indications for their use and avoid complications. We believe that selecting the right NMBA, administering concomitant sedation and analgesic therapy, and using appropriate monitoring techniques mitigate these risks for critically ill patients. Therefore, we review the indications of NMBA use in the critical care setting and discuss the most appropriate use of NMBAs in the intensive care setting based on their structure, mechanism of action, side effects, and recognized clinical indications. Lastly, we highlight the available pharmacologic antagonists, strategies for sedation, newer neuromuscular monitoring techniques, and potential complications related to the use of NMBAs in the ICU setting. The introduction of neuromuscular blocking agents to the ICU provides intensivists a unique capability in the management of critically ill patients. As with any therapy, however, the use of NMBAs has inherent risks, particularly when providers are unfamiliar with the nuances of selecting the appropriate agent, monitoring the depth of neuromuscular blockade, and ensuring adequate skeletal muscle recovery once NMBA therapy has ceased. Optimal neuromuscular blockade management has challenged clinicians for decades, despite the frequent use of NMBAs in clinical practice . ComplicWhile guidelines can help clinicians navigate many clinical scenarios, these recommendations are often limited by the lack of well-designed prospective trials. Ultimately, a thorough understanding of neuromuscular blockade management can equip clinicians to deal with scenarios that fall outside of the scope of medical specialty guidelines. This review provides up-to-date evidence to aid clinicians in selecting the right scenarios for establishing neuromuscular blockade in the ICU as well as choosing the optimal agent for such scenarios. Additionally, we will review methods to determine the level of neuromuscular blockade, the use of NMBA antagonists, and the optimal methods to confirm an adequate neuromuscular recovery and avoid prolonged residual weakness in this vulnerable patient population.In 2016, a task force comprising 17 members from the Society of Critical Care Medicine (SCCM) proposed updated and comprehensive recommendations for the use of neuromuscular blocking agents in the critically ill patient , and the risk of a “failed intubation” is several-fold greater in the ICU . Unlike 95]) can have a similar mean onset time , a characteristic that makes this agent suitable for rapid sequence induction and intubation (RSII) .Ta.Ta25].2/FiO2) ratio less than 150 . Indeed, patients with acute respiratory distress syndrome (ARDS) are unlikely to oxygenate or ventilate optimally with sedation/analgesia regimens alone. Gainnier et al. conducted a multi-center, prospective controlled randomized trial and found that the use of NMBAs during a 48-h period in ARDS patients was associated with a sustained improvement in oxygenation [In the ICU, NMBAs are also commonly used for the facilitation of mechanical ventilation. The current SCCM clinical practice guidelines  suggest genation . In the genation . Howevergenation . While cgenation , its cligenation –32. Thusweak recommendation with very low quality of evidence). Interestingly, more recent investigations have suggested that replacing neuromuscular blockade with continuous deep sedation regimens did not change the incidence of muscle weakness in this group of patients, suggesting that prolonged immobilization and inactivity are key clinical contributors to this complication rather than solely due to the administration of NMBAs [The neuromuscular blockade has been used in patients with status asthmaticus. However, this specific application’s use has decreased over concerns of severe weakness and critical care myopathy –35. Indeof NMBAs .insufficient evidence). Neuromuscular blockade may be useful in the short-term without negatively impacting hemodynamic parameters such as ICP, cerebral perfusion pressure (CPP), and blood pressure [In patients with an acute brain injury, a mass occupying lesion or subsequent intracranial edema, increases in cerebral perfusion can cause a deleterious increase in intracranial pressure (ICP). However, the current SCCM clinical practice guidelines  could nopressure . Furtherpressure , 37. TheAs in ARDS, the early use of NMBAs in sepsis may reduce in-hospital mortality , 39. Curinsufficient evidence). A complication from hypothermia is shivering, which leads to the deleterious consequences of increased metabolic rate and ICP, heat production, inflammation, and decreased brain tissue oxygen levels [weak recommendation, very low quality of evidence).In patients who suffer an out of hospital cardiac arrest, the use of therapeutic hypothermia plays an important role in survival to discharge . Howevern levels . The Amen levels , 43. IndThe only neuromuscular blockade patient management recommendation that was rated as “strong” by the SCCM panel of experts was the use of lubricating drops or gel along with eyelid closure for patients receiving continuous infusions of NMBAs . AdditioNMBAs cause skeletal muscle relaxation by blocking the transmission of impulses at the neuromuscular junction (NMJ) . These a95) can cause clinically relevant histamine release, producing tachycardia, hypotension, and skin flushing [Atracurium is an intermediate-acting NMBA that is metabolized through nonspecific plasma esterase-mediated hydrolysis as well as Hofmann elimination reaction in which the compound is degraded based on body pH and temperature . This brflushing .95). This intermediate-acting agent is also metabolized through organ-independent mechanisms via the Hofmann elimination reaction, making this benzylisoquinolinium drug one of the most commonly utilized NMBAs in critically ill patients who require neuromuscular blockade [Cisatracurium is the cis-cis isomer of atracurium, a feature that increases its potency four-fold, without the associated histamine release; therefore, a smaller dose is required for tracheal intubation ; patients deficient in this enzyme can have prolonged effects [Unlike cisatracurium and atracurium, mivacurium is a short-acting nondepolarizing NMBA. Mivacurium was developed in the 1990s and has recently been reintroduced to the US market . Antagon effects .95) produces a similar average onset time to that of succinylcholine, although individual patient responses can vary [Rocuronium is an intermediate-acting NMBA and is the only nondepolarizing drug that is currently utilized in a rapid sequence induction and intubation. A dose of 1.2 mg/kg  at the postsynaptic nicotinic receptors. Following the administration, succinylcholine produces a reliably rapid blockade and can be used to facilitate rapid sequence induction and tracheal intubation. Its use is associated with skeletal muscle fasciculations after administration, and waiting at least 30 s after the cessation of fasciculations should provide optimal blockade for endotracheal intubation , 69. SucIn the perioperative setting, pharmacologic antagonism of neuromuscular blockade is routinely used to restore baseline function and reduce the risk of postoperative residual paralysis . CurrentNeostigmine and edrophonium antagonize the action of NMBAs by preventing the action of the enzyme acetylcholinesterase. This enzyme breaks down ACh in the neuromuscular junction, and its inhibition results in the accumulation of ACh that competes with NMBA for binding sites on postsynaptic receptors. Neostigmine should not be utilized to reverse moderate levels of neuromuscular blockade (train-of-four count < 1–3) but should be reserved for situations with the train-of-four count > 3 Table . Median -1; however, several studies have reported its use in patients with a significant renal disease without complications [Rocuronium and vecuronium can be antagonized with sugammadex, a gamma-cyclodextrin compound that encapsulates and binds these NMBAs. This encapsulation process occurs in the plasma, creating a concentration gradient that facilitates the transfer of aminosteroidal NMBA from the neuromuscular junction back into the circulation. The tightly bound, inactive sugammadex-aminosteroidal complex is then excreted in the urine . Sugammaications –81. In aications . Concernications ; howeverications . While nications  and its ications  and promications . In an eTitrating appropriate levels of neuromuscular blockade may be essential to avoid prolonged paralysis in the ICU . While tSeveral obstacles and limitations exist when utilizing a PNS. Significant inter-observer variability can exist when using a PNS as the providers may visually or tactilely evaluate the response to train-of-four stimulation . DiffereWhile not common practice, handheld quantitative (objective) monitoring technology is expanding and improving. The use of these devices is increasing in the perioperative arena, and their application to guide administration of NMBAs and confirm recovery from neuromuscular blockade perioperatively has recently been recommended by a panel of experts . QuantitQuantitative monitors can be categorized based on the mechanism by which the train-of-four count and/or ratio are measured . AccelerA comprehensive review of sedation strategies in the ICU is beyond the scope of this review. Nonetheless, vigilance is warranted in maintaining adequate sedation when NMBAs are utilized in order to avoid unintended patient awareness and recall. Clinicians must recognize markers of inadequate sedation such as tachycardia, hypertension, diaphoresis, and ventilator dyssynchrony. While the use of processed electroencephalography (EEG) has been shown to decrease the risk of intraoperative awareness in high-risk surgical patients , currentThe use of NMBAs in the ICU setting risks numerous complications. Most notably, neuromuscular blockade results in prolonged patient immobility that can lead to the development of acquired weakness, myopathy, pressure ulcers, nerve injuries, and risk of deep venous thrombosis (DVT) . BecauseMultiple studies have shown that there is a correlation between ICU-acquired weakness (ICUAW) and neuromuscular blockade , 105; hoUnintended  awareness and recall are also a major concern during the use of NMBAs , 111. InOnce patients’ tracheas are extubated, the most feared complication is hypoxemia and the subsequent need for reintubation. NMBAs have been known to cause adverse pulmonary outcomes  such as While the administration of NMBAs can prove to be a life-saving therapy in select critically ill patients, these medications have unique inherent risks as well. However, by understanding the pharmacology, dosing, drug interactions, side effects, and monitoring techniques, clinicians can safely maximize the benefits. As there are few prospective studies that support improved long-term outcomes for patients in the ICU, the administration of NMBAs should be limited to facilitating endotracheal intubation, prevention of shivering following therapeutic hypothermia, and avoiding increases in intracranial pressure in patients at risk associated with coughing or ventilator dysynchrony. Moreover, residual weakness following the use of NMBAs in the ICU is a particular concern, given this vulnerable population. This complication may occur more frequently in the ICU, given the abundance of patients with significant organ dysfunction and delayed drug (NMBA) elimination. We recommend continuous vigilance when NMBAs are used in critically ill patients, selecting the most appropriate NMBA for each individual clinical scenario, evidence-based protocols that ensure adequate sedation and analgesia, appropriate equipment for assessing the degree of neuromuscular blockade, and aggressive physical therapy regimens during periods of reduced mobility. Such a multifaceted approach can improve patient safety when NMBAs are utilized in the ICU and reduce associated complications.


DNA vaccines offer a flexible and versatile platform to treat innumerable diseases due to the ease of manipulating vaccine targets simply by altering the gene sequences encoded in the plasmid DNA delivered. The DNA vaccines elicit potent humoral and cell-mediated responses and provide a promising method for treating rapidly mutating and evasive diseases such as cancer and human immunodeficiency viruses. Although this vaccine technology has been available for decades, there is no DNA vaccine that has been used in bed-side application to date. The main challenge that hinders the progress of DNA vaccines and limits their clinical application is the delivery hurdles to targeted immune cells, which obstructs the stimulation of robust antigen-specific immune responses in humans. In this updated review, we discuss various nanodelivery systems that improve DNA vaccine technologies to enhance the immunological response against target diseases. We also provide possible perspectives on how we can bring this exciting vaccine technology to bedside applications. The development of new vaccines to address emerging diseases continues to be of great importance and a major focus of medical research. Traditional vaccines which include live-attenuated, inactivated, and subunit vaccines have historically been effective in treating infectious diseases . HoweverDNA vaccine technologies present a promising alternative vaccine platform that has the potential to treat many diseases, such as cancers, atherosclerosis, and diabetes . DNA vacDespite years of research, DNA vaccine technologies have had limited success in the clinic and none have been approved for human use. The weak immune response associated with low gene transfection of DNA vaccines is one of the major associated challenges . Thus, tIn order to achieve successful gene transfection and stimulate the production of exogenous proteins, DNA vaccines must overcome a large number of extracellular and intracellular barriers. For intravenous delivery, DNA vaccines must be protected from degradation by DNase and phagocytic elimination through the reticuloendothelial system (RES) . FurtherSeveral technologies exist that can facilitate the delivery of plasmid DNA to cells to potentiate immune responses. Electroporation is one such technology that involves delivering electrical pulses to induce temporary and reversible permeability of the cell membranes. This, in turn, facilitates transient and stable cellular entry of DNA molecules . AlthougAlthough mechanical delivery methods for DNA have been shown to be effective, they suffer from the need for expensive equipment, excessive cellular damage, or both. For this reason, non-mechanical delivery methods are highly favored. Since high transfection efficiency is crucial for DNA vaccines to be effective, viral delivery vectors are most prominent. Millennia of natural selection enabled the evolution of efficient viruses capable of subverting the human body’s defense mechanisms, resulting in highly effective gene transporters . These sVaccines can be administered a number of ways including oral, intramuscular, intravenous, intradermal, intranasal, topical, and intratumoral. Based on the target tissue and mode of administration, the material composition and chemistry of the DNA nanodelivery system can be designed and tuned to improve therapeutic efficacy for specific environment and applications . WhetherNanoparticles (NPs) implemented as delivery vehicles provide protection of the payload, whether that be vaccine antigens, proteins, drugs, or nucleic acids, against degradation from harsh environmental conditions encountered during transportation to target cells . NPs areco-glycolic acid) (PLGA) [Polymer NPs are amongst the most widely investigated materials for nucleic acid delivery platforms due to their favorable safety profile, versatility, and ability to enhance immune responses ,28,29,30) (PLGA) ,27. The Mycobacterium tuberculosis was developed using mannosylated chitosan NPs as the delivery vector [Chitosan is an abundant polysaccharide that is a biocompatible, biodegradable, minimally toxic polymer suitable for biomedical applications ,30,31. Sy vector . The resy vector . Specifiy vector . In anoty vector . Chitosay vector .co-glycolic acid) (PLGA) is one of the most widely used polymer materials for drug delivery systems largely due to its FDA approval and enabled by its biodegradability, biocompatibility, and easily tunable physical properties [Streptococcus agalactiae [Mycobacterium tuberculosis latency antigen, as a primer prior to administration of a Rv1733c protein boost [Poly, and complexed with TRP-2 pDNA to form nanocomplexes for microneedle-assisted transcutaneous immunization [1800-Man/pTRP-2 promoted TRP-2-specific cellular immune responses, resulting in potent tumor growth inhibition and prolonged survival time of B16-xenografted mice. In this study, the solid microneedles served as a penetration enhancer by breaching the stratum corneum, a layer of the skin that functions as a physical barrier against the external environment which has proven to be a significant barrier for intradermal vaccination. In another recent study, an intradermal pH1N1 DNA vaccine delivery system was developed using microneedles coated with polyplexes containing PLGA/PEI NPs as shown in Polyethylenimine (PEI) is versatile with material properties and behavior that varies greatly with molecular weight and the degree of branching . High monization ,60. The Poly(ethylene glycol) (PEG) is an FDA approved polymer commonly used to surface functionalize NPs . The priThe studies detailed above demonstrated that polymeric nanomaterials offer a wide array of functionality attributed to their versatility in regard to their composition and molecular weight. Polymers offer good biocompatibility and safety profiles, positioning these materials as great candidates for the design of non-viral delivery vectors for DNA vaccines. Despite the generally safe nature of polymer-based delivery systems, the limited transfection efficiency they provide restricts their wide spread adoption and progression in clinical trials and beyond. As polymer science research continues to advance, non-viral and polymer-based DNA vaccine systems will continue to improve and eventually will provide comparable transfection performance to viral vectors that are the current standard in vaccine technology.For DNA vaccines as well as general nucleic acid delivery applications, lipid-based NPs are another prominent class of material used as non-viral vectors . LiposomPairing liposomes’ inherent properties with specific surface ligands can create potent targeted delivery platforms. Liposome NPs can be functionalized with shikimic acid, a molecule similar to mannose, capable of binding to mannose receptors and enabling targeted delivery to DCs . In one Lipid-DNA NP complexes have also been used as vaccine adjuvants. Lipid NPs complexed with non-coding plasmid DNA were implemented as an adjuvant for a whole-inactivated influenza A virus (IAV) vaccine in rhesus macaques. The results demonstrated the ability of the lipoplex adjuvant to stimulate increased levels of general IAV nucleoprotein and matrix-1 protein-specific antibodies in addition to greater activation of natural killer (NK) cells. In conjunction with the whole-inactivated IAV vaccine, the lipoplex adjuvant was shown to suppress uncontrolled viral replication and reduce levels of viral RNA in vivo .Various other lipid-based particles have been investigated for DNA vaccine delivery applications in order to overcome the weaknesses associated with liposome-based systems. One such material is the niosome, consisting of cholesterol or cholesterol-like molecules and non-ionic surfactants which form a highly stable bilayer vesicle through protection against lipid oxidation ,74. The Recently in 2018, a lipid NP system for small interfering ribonucleic acid (siRNA) delivery to treat Transthyretin amyloidosis, a peripheral nerve disease, was approved by the FDA . This waIn the previous sections, the strengths and weaknesses of both polymer-based delivery systems and lipid-based delivery systems were discussed. Polymeric materials offer highly biocompatible, biodegradable, and non-immunogenic properties with the capacity to be easily modified for functionalization to increase stability, and provide controlled release, and targeted delivery. However, polymeric gene delivery vectors generally suffer from poor transfection efficiency and the inability to produce significant exogenous protein expression. Conversely, lipid-based delivery systems offer high transfection efficiency and biocompatibility, however, they are hindered by their large size, toxicity, structural instability, and rapid systemic clearance. Combining these two technologies unlocks the ability to harness the advantages of both polymeric and lipid materials for nucleic acid vaccine delivery while compensating for the opposing material’s weaknesses. Such a delivery system is called a lipopolyplex which consists of a polymer-nucleic acid complexed core encapsulated by a liposome shell. Often the liposome shell is decorated with an outer lipid-PEG layer which functions as a steric stabilizer, preventing immune detection and enhancing the systemic circulation time. The lipopolyplex design provides the synergistic benefits of both polymer and liposomal materials, offering higher transfection efficiency and lower cytotoxicity than other platforms implementing only one or the other ,83.In an effort to develop an efficient DNA vaccine delivery system targeting the Peyer patch, plain liposomes were compared to polymer-liposome hybrid NPs. It was demonstrated that oral administration of the chitosan-coated lipopolyplex design was able to generate relevant levels of exogenous green fluorescence protein (GFP) expression throughout the intestine, while a plain chitosan-coated liposome was only able to generate exogenous GFP expression within the upper duodenum. The results showed that the lipopolyplex design induced a prolonged systemic circulation time through decreased enzyme degradation and was, in general, more effective in delivering DNA to target tissue than the non-hybrid NP counterpart . This prLipid-polymer hybrid NPs offer the ability to combine the advantages of both polymer and lipid materials in a single design. Although there is comprehensive research in the development of new materials for efficient DNA delivery, there is a lack of informative studies comparing existing delivery technologies. As such, it is difficult to understand whether polymer NPs or lipid NPs are more effective for DNA vaccination, let alone hybrid systems comprising of the two. To further improve the hybrid NP systems for DNA delivery, comparative studies of different delivery systems should be conducted, and this would provide further understanding to investigate what combination of materials, in terms of composition and proportion, are optimal for the delivery of DNA vaccine.Inorganic nanomaterials have attracted significant interest in DNA vaccine delivery applications due to their ease of functionalization, biocompatibility, and well-defined chemistry. Moreover, their thermal and chemical stability facilitates the process of sterilization, which is not possible with other classes of materials . InherenAmong the inorganic nanomaterials, gold (Au) NPs are a widely-used material due to their well-defined and versatile surface chemistries, ease of fabrication, and good biocompatibility. They play an important role in the field of vaccination as both a delivery platform, and an adjuvant, capable of reducing toxicity, enhancing immunogenic activity, and providing stability in regard to vaccine storage. A considerable number of papers have been published in the past two decades that are devoted to the use of gold NPs for generating DNA vaccines ,99,100. Ferric NPs are another class of materials that has been widely implemented as gene delivery vectors due to their favorable properties including low toxicity, inexpensive cost, ease of surface functionalization, and the ability to bind biological materials. Ferric materials can also provide unique magnetic properties that have been exploited for applications in imaging , tumor a2 NPs as a delivery vehicle for a Newcastle disease virus (NDV) DNA vaccine, encoding F gene plasmid DNA (pFDNA). Intranasal immunization of pFDNA-Ag-SiO2 NPs was capable of sustained release of the vaccine, inducing strong humoral and cellular immunity [2-LDH NPs not only induced much higher serum antibody responses than naked plasmid DNA, but also promoted T-cell proliferation and skewed T helper cells towards Th1 polarization.Other types of nanomaterials such as silver (Ag) NPs, layered double hydroxide (LDH) NPs, and calcium phosphate NPs have also been used as DNA vaccine delivery vectors and adjuvants ,112,113.immunity . In otheimmunity . When coVirus-like particles (VLPs) have been developed to circumvent the problems associated with conventional virus-based vaccination strategies including immunogenicity, reversion to virulence and insertional mutagenesis . VLPs arFullerenols, or polyhydroxy fullerenes, are capable of self-assembling into virus-like particles with an average size of 40 nm, designed with dual functionality. First, as a nano-adjuvant capable of enhancing immune responses to a targeted vaccine, and second, as a high-loading capacity DNA delivery vector. Fullerenols have been studied as a proof-of-concept DNA vaccine delivery system, using HIV-1 DNA antigen as a model antigen for immunization in mice . In vitrVLPs from different origins differ in their stability, therefore the shelf life of different VLPs needs to be studied to get a comprehensive understanding of the materials used . The NPsPeptide-based nanocarriers are a favorable platform for DNA vaccine delivery due to their inherent biocompatibility, biodegradability, and generally low cytotoxicity ,124. AltFor gene therapy applications in general, an extremely well-studied protein for nucleic acid delivery is protamine. Protamine is a cationic protein whose structure efficiently complexes plasmid DNA and possesses several nuclear localization signaling regions that mediate transport across the nuclear membrane. Thus unsurprisingly, protamine is a popular delivery platform for DNA vaccines. An additional appeal of protamine as a DNA delivery vector is that protamine sulfate has been approved by the FDA to reverse the effects of heparin, thereby being a safe and convenient material for the development of new vaccines. In one study, mannosylated protamine sulfate (MPS) was complexed with plasmid DNA encoding gastrin-releasing peptides (GRP). GRP is an autocrine growth factor that can stimulate tumor progression in certain cancers when interacting with GRP receptors and are thus a potential target for cancer immunotherapy. The results of the study demonstrated that the mannose functionalization of the protamine particles resulted in higher transfection efficiency in APCs compared to the non-mannose-functionalized counterparts. In vivo studies demonstrated that the MPS-GRP system induced significant levels of anti-GRP antibodies, preventing GRP binding to GRP receptors and resulting in effective tumor growth inhibition . In anotRecently, a peptide-based DNA vaccine nanovector was developed which induced enhanced cellular and humoral immune responses against HIV via intramuscular, intradermal, and subcutaneous injection . Four amProtein-based delivery systems have seen limited use for DNA delivery specifically due to their low loading capacity, which restricts their overall therapeutic efficacy. For the same reason, greater success with siRNA delivery has been achieved because they are much smaller in size, enabling higher concentrations of loading, and enabling greater therapeutic effect. Despite these challenges, further research should be continued to improve the loading capacity of DNA in proteins, because they offer the unique ability to serve a dual purpose of binding and delivering DNA, but also providing additional functionality, either acting as an antigen or adjuvant itself, or mediating transport across cell membranes or nuclear membranes.Based on existing literature, there have been only a few nanomaterial-based DNA vaccine delivery systems that have successfully progressed to clinical trials, and none have been approved for use thus far . ResultsOne method to address this challenge is through the use of adjuvants. Similar to inactivated and subunit protein-based vaccines, DNA vaccines can also benefit from the use of adjuvants to enhance the protective immune responses generated. One nanomaterial-based adjuvant that was studied in a clinical setting is Vaxfectin, a cationic liposome that can ionically bind to DNA and potentiate the immune response against H5N1 influenza-associated proteins including HA, nucleoproteins, and viroporins ,123. ThiAnother major application of DNA vaccine technology is for the use of cancer immunotherapy. Clinical studies in the past have demonstrated the safety of DNA vaccination in humans, showing minimal adverse reactions and the ability to induce a wide range of specific immune responses, however, the therapeutic effects have been minimal which has impeded the progress of this vaccine technology. The primary reason for low immunogenicity of DNA vaccines in humans is due to inherent immunosuppressive properties of the tumor microenvironment, including tumor epitope mutation, T cell exhaustion, antigen tolerance, infiltration of immunosuppressive cells and tumor-associated macrophages that produce immune-suppressing microenvironments  . There aDespite the optimistic advancements in the past decade of vaccine research, there are still no commercially available vaccines or effective treatments for many diseases such as cancer, HIV, dengue, zika, and chikungunya. DNA vaccines pose as promising candidates to fill this gap due to the ability to change the vaccine targets simply by changing the protein encoded within the DNA delivered. The main challenge associated with DNA vaccines is generating therapeutically relevant levels of exogenous protein expression within APCs in order to stimulate protective humoral and cell-mediated immunity. The two main methods through which this can be achieved is the use of DNA delivery vectors and immune response enhancing adjuvants. Nanomaterials have been widely researched for use as DNA delivery vectors and adjuvants due to their wide range of properties that can be tuned through surface functionalization. Polymer, lipid, metallic, and inorganic materials have a diverse set of properties with unique advantages and weaknesses, all of which have been seminal contributions in the field of DNA vaccine research, resulting in improved efficacy and more favorable safety profiles.Despite these advancements in nanomaterial-based delivery systems, DNA vaccines have still performed poorly in human clinical trials to date, failing to generate potent immunogenic responses ,134. OneSince one of the major challenges with DNA vaccines is low immunogenic responses, several other factors including the selection of the encoded antigen, combination therapy, and a treatment schedule can be considered to improve their efficacy in clinical trials. Specifically, decisions such as which antigens should be selected to be encoded within the DNA payload to maximize the effectiveness of the vaccine, as well as how many antigens should be encoded, are crucial details which we do not currently have the knowledge to optimize. Knowing which antigens induce more potent and therapeutically relevant immune responses are important criteria that are often not discussed in preclinical studies which is a major detriment to their clinical success. Moreover, it is unknown what combination of antigens maximizes therapeutic efficacy of the vaccine, and whether antigens should be encoded within the same plasmid DNA molecule, or whether it should be encoded in a separate molecule and administered together. Similarly, it is difficult to predict which adjuvants and co-stimulatory molecules should be used to optimize the effects of the DNA vaccines. The rationale behind selecting one combinatorial treatment compared to another is often overlooked in both preclinical and clinical studies, which presumably limits the efficacy of the DNA vaccines in those study settings. To address many of these issues, more investigative research is required to understand the current capabilities of existing technologies, and the hierarchies of effectiveness. This requires more comparative studies, as opposed to most studies that report on novel designs and systems. Specifically for NP-based DNA vaccines, the major challenges that hinder their translation to the clinic are common issues that affect NP-based drug delivery technologies in general . Most si


Purpose: Pubertal suppression is standard of care for early pubertal transgender youth to prevent the development of undesired and distressing secondary sex characteristics incongruent with gender identity. Preliminary evidence suggests pubertal suppression improves mental health functioning. Given the widespread changes in brain and cognition that occur during puberty, a critical question is whether this treatment impacts neurodevelopment.Methods: A Delphi consensus procedure engaged 24 international experts in neurodevelopment, gender development, puberty/adolescence, neuroendocrinology, and statistics/psychometrics to identify priority research methodologies to address the empirical question: is pubertal suppression treatment associated with real-world neurocognitive sequelae? Recommended study approaches reaching 80% consensus were included in the consensus parameter.Results: The Delphi procedure identified 160 initial expert recommendations, 44 of which ultimately achieved consensus. Consensus study design elements include the following: a minimum of three measurement time points, pubertal staging at baseline, statistical modeling of sex in analyses, use of analytic approaches that account for heterogeneity, and use of multiple comparison groups to minimize the limitations of any one group. Consensus study comparison groups include untreated transgender youth matched on pubertal stage, cisgender  youth matched on pubertal stage, and an independent sample from a large-scale youth development database. The consensus domains for assessment includes: mental health, executive function/cognitive control, and social awareness/functioning.Conclusion: An international interdisciplinary team of experts achieved consensus around primary methods and domains for assessing neurodevelopmental effects  of pubertal suppression treatment in transgender youth. When administered in early puberty, GnRHa suppress endogenous sex hormone production and prevent the development of undesired and irreversible secondary sex characteristics, thereby minimizing distress associated with pubertal development incongruent with gender identity.5 For youth who later decide to initiate estrogen/testosterone (gender-affirming hormones [GAH]) treatment to induce development of the desired secondary sex characteristics, pubertal suppression may minimize the need for more invasive, surgical interventions . For youth who decide not to pursue GAH treatment, discontinuing GnRHa will reactivate the hypothalamic-pituitary-gonadal axis and endogenous puberty will resume.6Standards of care established by the World Professional Association for Transgender HealthN=70)7 and later in early adulthood after surgery for gender affirmation (N=55).8 The third study compared groups of GnRHa-treated (n=35) and untreated (n=36) youth longitudinally.9 Findings across these studies include significant reductions in depressive symptoms and improvement in overall psychosocial functioning in GnRHa-treated transgender youth. A fourth cross-sectional study compared adolescents diagnosed with gender dysphoria (GD), who were treated with GnRHa and close to starting GAH treatment (n=178), adolescents newly referred for GD evaluation (n=272), and cisgender adolescents recruited from the general population (n=651) on self-reported internalizing/externalizing problems, self-harm/suicidality, and peer relationships.10 Before medical treatment, clinic-referred adolescents reported more internalizing problems and self-harm/suicidality and poorer peer relationships compared to age-equivalent peers. GnRHa-treated transgender adolescents had fewer emotional and behavioral problems than clinic-referred, untreated adolescents and had comparable or better psychosocial functioning than same-age cisgender peers. In addition to studies of youth, the 2015 U.S. Transgender Survey included questions about past gender-affirming medical treatment, including pubertal suppression. These questions were asked retrospectively and linked to reported current and lifetime mental health.11 Individuals who received pubertal suppression treatment (n=89), when compared to those who wanted pubertal suppression, but did not receive it (n=3405), had lower odds of endorsing lifetime suicidal ideation on the survey. Given these five studies and the presumed reversibility of GnRHa treatment, pubertal suppression is increasingly offered to early pubertal transgender youth. It is important to note that there has been only one longitudinal report of adult outcomes,8 and questions remain regarding the potential for both positive and disruptive effects of pubertal suppression on neurodevelopment.12–14Three longitudinal studies have examined psychosocial outcomes in GnRHa-treated transgender youth; two (conducted by the same research group) followed a single cohort over time, immediately before initiating GAH ,17 and social thinking and competence.19 During this period, there is a developmental shift toward greater exploration and novelty seeking,21 salience of peer perspectives and interactions,22 and accelerated development of passions/interests and identities.23 These developments lay the groundwork for adult functioning.24 At the level of the brain, several primary neurodevelopmental processes unfold during adolescence, including myelin development25 and changes in neural connectivity26; synaptic pruning27 and gray matter maturation29; changes in functional connectivity30; and maturation of the prefrontal cortex31 and the “social brain” network.19 Adolescent neurodevelopmental processes underlie mental health risks, resilience, and outcomes.33The pubertal and adolescent period is associated with profound neurodevelopment, including trajectories of increasing capacities for abstraction and logical thinking,34 Androgen and estrogen receptors are found in high density within the hypothalamus and amygdala, and are also present in the hippocampus, midbrain, cerebellum, and cerebral cortex of the rodent and monkey.35–37 This widespread receptor distribution in rodents may explain the diverse effects of pubertal hormones on both reproductive and nonreproductive behaviors, including anxiety, scent-marking, and food guarding.34 In human studies, pubertal progression has been linked to developmental changes in reward,38 social,39 and emotional processing40 as well as cognitive/emotional control.41 However, consensus regarding pubertal impacts at the neural level—such as puberty-associated changes observed in magnetic resonance imaging (MRI) measures—has been more difficult to achieve.42 Distinct puberty-related neurodevelopmental trajectories have been differentiated by sex.43Considerable research has addressed the effects of puberty-related hormones on neurodevelopment, including hormone manipulation studies in nonhuman animals and observational studies in humans. Animal studies demonstrate pubertal hormones exert broad neuronal influence, including effects on neurogenesis, differentiation, apoptosis, dendritic branching, spine density, and regional gray and white matter volumes.sensitive period for brain organization:44–46 that is, a limited phase when developing neural connections are uniquely shaped by hormonal and experiential factors, with potentially lifelong consequences for cognitive and emotional health. Studies have linked early life adversity to early puberty onset47 and early puberty onset to poorer mental health.48 There is also some evidence to suggest that delayed puberty onset predicts slightly poorer adult functional outcomes.49 Taken as a whole, the existing knowledge about puberty and the brain raises the possibility that suppressing sex hormone production during this period could alter neurodevelopment in complex ways—not all of which may be beneficial.The combination of animal neurobehavioral research and human behavior studies supports the notion that puberty may be a 50 No group differences were found in task load-related brain activation; GnRHa-treated transgender girls demonstrated poorer performance compared with untreated transgender boys and cisgender controls. Schneider et al. evaluated a single pubertal transgender girl undergoing GnRHa with MRI scans of white matter and cognitive assessments at baseline (before GnRHa initiation) and at 22 and 28 months of pubertal suppression treatment.51 During follow-up, white matter fractional anisotropy  did not increase in the manner otherwise expected during puberty. By 22 months of pubertal suppression treatment, working memory scores dropped by more than half a standard deviation.Two small studies have assessed impacts of pubertal suppression on neural and cognitive functioning in peripubertal transgender youth. Staphorsius et al. compared brain and behavioral responses of GnRHa-treated  and untreated transgender youth (10 of each sex) during an executive function task.52 In sum, GnRHa treatment might produce a myriad of varied impacts, both positive and disruptive.Larger-scale, longitudinal studies are required to understand possible neurodevelopmental impacts of pubertal suppression over time in transgender youth. Suppressing puberty may reduce dysphoria and diminish risks for poor mental health in this population, thereby exerting neuroprotective effects. If pubertal suppression disrupts aspects of neurodevelopment, it is possible these effects are temporary, with youth “catching up” developmentally after transitioning to GAH treatment or discontinuing GnRHa. However, pubertal suppression may prevent key aspects of development during a sensitive period of brain organization. Neurodevelopmental impacts might emerge over time, akin to the “late effects” cognitive findings associated with certain oncology treatments.The goal of this study was to develop a framework in which these questions could be asked, and ultimately answered. We identify priority research methodologies that can be used to address the empirical question of how pubertal suppression in transgender youth may affect neurodevelopment and real-world functioning. Given the complexity of neural development during the pubertal period and the novelty of developmental research with transgender youth, this study employed a Delphi consensus method to leverage international expertise in neurodevelopment, gender development, puberty/adolescence, neuroendocrinology, and statistics/psychometrics. By engaging a community of experts in an iterative consensus-building procedure, this study aimed to advance thinking about efficacious designs by moving beyond individual research efforts and single-discipline approaches.54 and has been used extensively to address health-related questions, particularly in emerging fields of clinical care.55–57 In the first round of a two-round Delphi procedure, a key question is presented to experts, who remain anonymous to one another throughout the Delphi process. Each expert provides responses/solutions to the question, which are then combined and organized by the study team. In the Delphi round two, experts rate each proposed statement/solution according to the level of agreement. Responses reaching the a priori consensus criterion are included as consensus statements. Given its anonymous iterative nature, the Delphi method avoids problems of typical expert work groups  and allows for interaction among larger groups of experts from diverse locations and disciplines through asynchronous communication.58–60The Delphi procedure is a reliable iterative research method for establishing expert agreement,What, if any, real-world impact does pubertal suppression have on transgender children's cognitive and neural development? International experts in relevant research fields were identified and invited as follows:1.An independent advisory panel consisting of five experts across key disciplines (see Acknowledgments section) was formed to identify international experts who, based on knowledge and experience, could best propose a research design to assess neurodevelopmental impacts of pubertal suppression in transgender youth.2.Thirty-two recommended experts were vetted for their expertise; all met required criteria .3.These experts were invited to participate in the Delphi procedure and were informed they would be invited to consider being a co-author of the resulting article. Twenty-eight experts responded: 20 agreed to participate, 4 declined due to lack of time, and 4 declined due to self-reported lack of expertise in this research area. Snowball sampling identified an additional 16 recommended experts, who were vetted (as described above) for their experience. Eight met criteria and were invited. Five of these experts participated, yielding a total of 25 experts agreeing to participate, 24 of whom completed the Delphi process. See We employed a two-round Delphi procedure to obtain expert consensus regarding the most efficacious research design elements to address the following research question: The Ann & Robert H. Lurie Children's Hospital of Chicago Institutional Review Board found that an expert Delphi consensus initiative did not require informed consent since the experts were direct partners in the research product. The first round of Delphi survey was distributed through the REDCap online survey platform and presented an overview of the research question with the following prompt for respondents: “What methods and tools should we use to identify clinically meaningful neurodevelopmental impacts of pubertal suppression? What type of longitudinal design and follow-ups are both practical and appropriate? What comparison groups might we consider?” This initial process yielded 131 distinct research design considerations; multiple descriptions of the same concept were collapsed into single statements. In the second Delphi round, each first-round research design consideration was presented back to the experts and rated as follows: a priority idea/approach or not a priority idea/approach. Experts could also select, “cannot rate due to lack of expertise.” The first Delphi round also yielded lists of potential comparison groups and assessment domains (29 items). In the second Delphi round, participants were asked to rank order these items according to priority. For the priority rankings of comparison groups, the top-rated comparison group by each expert was given a value of 2 and the second rated comparison group was given a value of 1. A mean was calculated for each comparison group option based on these values and these mean scores were used to identify the overall priority rankings. For the list of priority domains to measure, a parallel approach was taken with the top 6 domains ranked by each expert.All experts participated in the second Delphi round. Twenty-two of the Delphi experts participated in the construction of the resulting article and are co-authors listed in reverse alphabetical order by last name (authors 5–26). The Four of the 131 individually presented statements were excluded from analyses because fewer than 15 experts rated them. Of the remaining 127 statements, 44 met the 80% or higher criterion for consensus and inclusion  there may be a larger database of norms available that the cohort could be compared to, in addition to a local comparison (control) group(s), (2) general composite scores within test batteries tend to provide more reliable and stable scores than individual tests, and (3) tasks within a category may be swapped in case of worries for learning effects. In any study of cognitive change based on serial assessments, reliability of measures is paramount (the consensus in the field is that tests should have a minimum test-retest reliability of >0.70). It may be pragmatic to use measures and methods from large representative studies, such as the Adolescent Brain Cognitive Development (ABCD) Study.All processes being studied  display considerable heterogeneity, and methods that fail to capture this will provide distorted findings and lead to biased clinical recommendations. Analyses based on group means  are unlikely to generalize to all individuals being treated. Therefore, it is necessary to collect enough data per person to characterize individual trajectories of change over time.per se. To the extent that pubertal suppression may also put some features of brain development on hold, it is critical to know whether these features “catch up” (either once GAH treatment is initiated or if the adolescent elects to stop GnRHa and resume endogenous puberty), or whether a sensitive window for hormone-dependent brain development has closed. One way to measure this is to assess whether neurodevelopment shifts in response to initiating GAH following pubertal suppression: Do GnRHa-treated youth stay on the same neurodevelopmental trajectory as when puberty was suspended or does this trajectory change?Any GnRHa-induced neurocognitive effect may be complicated by psychosocial and affective aspects of the transgender experience. Therefore, multivariate models of both cognitive and psychosocial functioning should be included. Accounting for differences between birth-assigned male youth versus birth-assigned female youth is important, as sex is differentially related to the rate and pattern of cognitive development, connectome distinctiveness, and timing of peak brain volume. Assessments should begin before puberty in both treatment and comparison groups. The effects of pubertal suppression may not appear for several years. Any GnRHa-related difference in brain structure is likely to be observed over the long term, rather than immediately. Shifts in social and affective learning processes might cause subtle short-term differences that could ultimately result in clinically impactful longer-term effects. Therefore, studies should follow GnRHa-treated youth over time, including the time period after GnRHa treatment ends and/or when GAH commence. Some aspects of human adolescent brain development are more related to pubertal hormone status than age To assess neurodevelopmental trajectories associated with GnRHa treatment, more than one comparison group is needed to minimize the limitations of any one comparison group. No single comparison group is ideal for this study question. A rank order of possible comparison groups is provided in 61 Studies in rodents also demonstrate that testosterone, acting during puberty, programs the ability to adapt behavior as a function of social experience.34 Measurement approaches should extend beyond cognitive capacities alone, embedding social, affective, and value-based learning processes. There is an emerging shift in thinking about increases in reward sensitivity and sensation-seeking during puberty as related to social-value learning.18 Dopamine release is not primarily a “reward” signal, but rather a learning signal —the natural increased salience of social learning . The effects of suspending puberty on the salience of social-value learning might produce small near-term effects, but could contribute to changes in patterns of behavior over time, leading to large individual differences in developmental trajectories for GnRHa-treated youth.It will be critical to prioritize assessment domains based on hypothesized mediating mechanisms, with the most important domains to measure as follows: mental/behavioral health, pubertal stage, executive function/control, gender identity/dysphoria, and social awareness/functioning. See 62 and is related to cognitive processing speed. Both structural MRI and diffusion tensor imaging approaches should be used for white matter imaging and analyzed using a longitudinal imaging pipeline for processing these data with scientific rigor.If neuroimaging is included, imaging approaches should focus on the following domains: social/emotional processing, executive functioning, risk and reward processing, and self-concept. Neuroimaging should parallel behavioral assessment. Neural measures should be linked to neurocognitive and behavioral measures. Acquisition parameters should be matched between imaging sites. Investigation of white matter development is important as myelination progresses during puberty, likely under the influence of sex hormones,Puberty suppression has become an increasingly available option for transgender youth, and its benefits have been noted, particularly in the area of mental health. However, puberty is a major developmental process and the full consequences  of suppressing endogenous puberty are not yet understood. The experts who participated in this procedure believe the effects of pubertal suppression warrant further study, and this Delphi consensus process develops a framework from which future research endeavors can be built.Expert consensus emphasized a minimum of three measurement time points, inclusion of multiple comparison groups to minimize the limitations of any one group, precision pubertal staging at baseline, accounting for sex in design and analysis, and the use of designs that capture heterogeneity in processes being studied. Focus on longer-term trajectories and outcomes was emphasized, given that effects of pubertal suppression on various processes may not be evident in the near term, and responses to delayed receipt of gonadal hormones may not be comparable to initial potentially organizing effects. Experts also highlighted that accounting for the psychosocial aspects of the transgender experience itself on development will require models that integrate both cognitive and psychosocial functioning. The highest endorsed measurement priorities were mental and behavioral health, executive function/cognitive control, and social awareness/functioning. The importance of interrelations between domains that mature during puberty/adolescence was also emphasized, including bidirectional relationships between cognitive and emotional control and links between reward sensitivity and social value learning. Regarding neuroimaging, experts stressed the importance of linking neural signatures to cognitive and behavioral measures, with attention to white matter development. Notably, while there was consensus in this approach to neuroimaging, there were divergent views as to whether a neuroimaging protocol should be prioritized in a study with limited resources. Some experts noted that insufficient work has been done on neural development during puberty in general and expending resources on an expensive neuroimaging protocol for this subset of youth may be premature, while others felt that defining underlying brain mechanisms by neuroimaging was important. Furthermore, at the final review of the article, four co-authors noted a concern with this specific Delphi consensus recommendation: “Accounting for differences between birth-assigned male youth versus birth-assigned female youth is important, as sex is differentially related to the rate and pattern of cognitive development, connectome distinctiveness, and timing of peak brain volume.” The four authors felt that instead of “peak brain volume,” a more appropriate measurement concept might be that of “structural brain metrics” .Twelve different comparison groups were proposed in the first round of the Delphi and 8 of the 12 groups were rated as either first or second priority by at least 1 expert in the second Delphi round. This heterogeneity underscores the complexity of selecting comparison groups for this research and lends support to the experts' recommendation to engage more than one comparison group. The highest rated comparison groups were untreated transgender youth matched on pubertal stage, cisgender youth matched on pubertal stage, and a sample from a large-scale quasi-normative database  used as a “brain growth curve.” These comparison groups are not without weaknesses. Untreated transgender youth may differ in their intensity or experience of GD, level of parent support , and socioeconomic status of the family and access to treatment . A cisgender comparison group would lack gender-minority experience and associated stress.Some statements approached, but did not reach consensus. For example, many experts suggested continuing assessments of transgender youth through young adulthood (mid-20s) when prefrontal development is near completion. Assessing adaptive functioning (everyday skills) over time due to the bidirectional link between executive functioning and adaptive behaviors was also often endorsed.63–66 The neurodevelopmental impacts of pubertal suppression on neurodiverse gender-diverse youth might well be different than in neurotypical gender-diverse youth, given variations in neurodevelopmental trajectories observed across neurodevelopmental conditions.67–69Not all relevant study considerations were raised by the Delphi panel. Neurodevelopmental impacts of pubertal suppression in transgender youth with neurodevelopmental differences/diagnoses  were not specifically addressed by the experts. Yet, evidence suggests an overoccurrence of neurodiversity characteristics  among gender-referred youth.per se. This reflects the dearth of transgender care clinicians/specialists with research productivity in adolescent neurodevelopment. Thus, the experts could comment with authority on neurodevelopment, including gender development/dysphoria aspects of study design, but the real-world clinical care considerations may well be underdeveloped in the proposed research design. For example, the everyday lived experience of transgender youth seeking gender-affirming medical care would be unfamiliar to most neurodevelopmental researchers. After the Delphi procedure was completed, one panelist commented that pubertal hormones might play a role in organizing neurodevelopmental gender-related trajectories, including identity itself, which would be important to consider for a developmental study of gender diverse youth.This study included experts from a range of relevant disciplines—a strength and also a possible limitation. The varied disciplines allowed for a broader range of ideas and perspectives, but some specialized recommendations might not have been sufficiently understood by Delphi experts from other disciplines. It is possible that some useful recommendations were lost in the process because few experts had backgrounds relevant to them. In fact, four recommendations were dropped from consideration because more than nine experts indicated they could not rate the item or skipped the item. These four items included topics related to advanced growth curve modeling, impact of GnRHa on immune system functioning, multifactorial relationships between GD and neurodevelopment, and challenges associated with using alternative forms of measures in longitudinal designs. The Delphi team included experts across the fields of neuroscience, neurodevelopment, developmental measurement, and gender development; however, most were not specialized in clinical transgender care Despite these limitations, an international expert team successfully completed an iterative Delphi procedure achieving consensus around priority research design elements to study neurodevelopmental impacts of pubertal suppression in transgender youth. The resulting consensus parameter addresses broad design issues, including comparison groups, longitudinal design, neurodevelopmental targets for assessment, and measurement approaches. While it may not be possible to incorporate all consensus methodologies into a single study, this parameter may serve as a roadmap for a range of research initiatives investigating pubertal suppression treatment in transgender youth.


Cancer therapeutics must be delivered to their targets for improving efficacy and reducing toxicity, though they encounter physiological barriers in the tumor microenvironment. They also face limitations associated with genetic instability and dynamic changes of surface proteins in cancer cells. Nanosized exosomes generated from the endosomal compartment, however, transfer their cargo to the recipient cells and mediate the intercellular communication, which affects malignancy progression, tumor immunity, and chemoresistance. In this review, we give an overview of exosomes’ biological aspects and therapeutic potential as diagnostic biomarkers and drug delivery vehicles for oncotherapy. Furthermore, we discuss whether exosomes could contribute to personalized cancer immunotherapy drug design as efficient nanocommunicators. Oncology drugs constitute the largest therapeutics section approved by the Center for Drug Evaluation and Research, a division of the United States Food and Drug Administration . Once caAnticancer drug approval trends have changed since cancer chemotherapeutic agents were first developed in the 1940s. Cancer chemotherapeutics show efficacy as well as side effects as they not only interfere with the growth or division of cancer cells but also normal cells. Cancer cells even evade anticancer drugs by mediating a cellular efflux of drugs or by reducing target gene expression. They not only receive external signals but also transmit signals to form new blood vessels in cancer tissues. EVs secreted by cancer cells transfer signals for cancer progression and metastasis .As technical limitations associated with cancer chemotherapy have been recognized, and the understanding of cancer biology has improved, the paradigm of research and development has shifted towards targeted therapy using monoclonal antibodies and small molecules that target the signaling process of cancer cells. Recently, synthetic small molecules such as a FGFR (fibroblast growth factor receptor) inhibitor , CSF1R (colony-stimulating factor-1 receptor)/RTK (receptor tyrosine kinase)/FLT3 (FMS-like tyrosine kinase 3) inhibitor , and exportin 1 inhibitor , and biologics such as an antibody-drug conjugate (ADC) targeting nectin-4 , and ADC targeting CD79B  have been approved. Interestingly, entrectinib (Genentech/Roche) simultaneously targets c-ROS oncogene 1 (ROS1), ALK (anaplastic lymphoma kinase) RTKs, and tropomyosin receptor kinase proteins encoded by neurotrophic tyrosine receptor kinase (NTRK) genes; it was approved as a biomarker-based treatment for ROS1-positive and NTRK fusion-positive cancer. In the same vein, engineered EVs that carry small interfering RNA (siRNA) or short hairpin RNA specifically targeting oncogenic KRAS mutation showed their therapeutic potential in pancreatic ductal adenocarcinoma mouse models .Despite the remarkable performance of targeted anticancer drugs, limitations have been associated with the targeting of markers on the cancer cell surface, because cancer cells are genetically unstable, and surface proteins in cancer cells change dynamically during disease progression . The necHowever, cancer therapeutics encounter barriers against transport to target sites owing to the elevated levels of solid stress, vascular network formation, interstitial fluid pressure, and density of extracellular matrix (ECM) in the tumor microenvironment . NanocarIn this review, we aimed to provide a comprehensive overview of the biological aspects and potential therapeutic applications of exosomes in cancer. Here, we also discuss whether exosomes could contribute to personalized cancer immunotherapy drug design as efficient nanocommunicators.Cells release EVs enclosed in lipid membranes into the extracellular environment. Exosomes, microvesicles (MVs)/microparticles, and apoptotic bodies form a subgroup of EVs. They have been defined by their biogenesis, size, or constituent molecules. The process of exosome biogenesis starts with endocytic membrane transport through which the cell surface proteins can be recycled . The perThe most well-known mechanism for packaging receptors internalized from the cell surface and other exosomal cargo proteins in the late endosome membrane depend on the endosomal sorting complex required for transport (ESCRT) machinery . CytosolAlternatively, ESCRT-independent pathways are supported by MVB formation even in the depletion of key subunits of ESCRTs . Sphingocis-acting elements that target RNAs to exosomes . Fi. Fi44]. Exosomes function as unique intercellular communicators and debris managers for cellular homeostasis . DeliverExosomes also regulate angiogenesis and vascular permeability . A mucin6β4 was associated with lung metastasis, whereas targeting the exosomal integrin αvβ5 was associated with liver metastasis. TEXs can also enter sentinel lymph nodes and influence lymph node distribution of cancer cells, which is driven by synchronized molecular signals that affect tumor metastasis . TE. TE92]. Exosomes are attractive nanovehicles for targeting cancer . As exosA number of studies have demonstrated that drug-loaded exosomes show better outcomes in inhibiting cancers, but the methods for loading drugs into exosomes also need to be explored further because they are closely related to the stability and loading efficiency of the drugs. To date, there are three types of drug loading methods for exosomes: exogenous loading, endogenous loading, and liposome fusion loading . ExogenoAs many of chemical drugs can act after being internalized into cancer cells, they need to diffuse through the cell membrane to exert cytotoxicity, which is one of the factors reducing the efficacy of drugs . In thisAs the efficacy of many proteins is limited due to several barriers such as short half-life, low delivery rate, and induction of resistance, the use of appropriate delivery vehicles is one of the best ways to achieve successful protein drug therapies. As exosomes can protect the contents from various enzymes and the immune system, they can act as effective delivery vehicles for proteins. Proteins can be loaded either inside or on the surface of the exosome, based on the mechanism of action of the drugs. However, as therapeutic proteins are macromolecules, it is difficult to directly incorporate them into exosomes. Therefore, genetic modification of the cells of origin leading to the expression of therapeutic proteins in exosomes is usually preferred to prepare protein-loaded exosomes. Several preclinical studies regarding protein-loaded exosomes are summarized in Similar to therapeutic proteins, delivery vehicles are an essential component of successful gene therapy. Exosomes can protect genes from various enzymes, such as DNases and RNases, and can also directly deliver genes inside the cells, which enhances their therapeutic efficacy. Different types of RNAs such as mRNA, miRNA, and siRNA are promising candidates for the treatment of cancers. Studies on exosome RNA delivery are summarized in Exosomes can be used as cell-free vaccines owing to the fact that exosomes derived from various donor cells, such as immune cells and cancer cells, are involved in fostering antitumor immunity . DEXs caBesides, CAR exosomes derived from effector CAR-T cells showed cytotoxic effects on cancer cells . IntraveRecently, cancer therapeutics has made great strides, and various clinical trials for targeted cancer therapy or immunotherapy have been conducted singly or in combination. However, there remains an unmet need, because only a few types of cancer patients are restrictedly responsive to current immune checkpoint blockers. In order to elevate response rate for personalized immunotherapy, prognostic biomarkers need to be established. As mentioned above, exosomal components can be used as diagnostic biomarkers in liquid biopsy, nanovehicles for delivery of anticancer drugs, and mediators between cells affecting tumor immunity. Detection of marker proteins or nucleic acids in circulating exosomes shows potential for predicting patients’ clinical response . “TEXs-oIn that vaccinating autologous exosomes obtained from patients can avoid allograft reaction and carry tumor antigens, patient-derived exosomes have received attention as good candidates for personalized immunotherapy. Using bone marrow aspirate from patients, MSCs are isolated and expanded ex vivo, and then large scale of MSCs engineered with anticancer genes can be transplanted to patients for personalized treatment . As exos