Clinical practice guidelines (CPGs) provide conveniently packaged evidence-based recommendations to inform clinical decisions. However, intended end-users often do not know how to source, appraise, interpret or choose among CPGs. Moreover, it can be confusing when recommendations on the same topic differ among CPGs, in wording, intent and underpinning evidence.This article reports on the processes of: (1) identifying current CPGs for acute and subacute low back pain (LBP) to fit the needs of South African physiotherapists, (2) collating and summarising CPG recommendations to produce a user-friendly end-user product and (3) testing the utility of the summary CPG document on South African physiotherapy clinicians to efficiently determine acceptability, appropriateness and feasibility to inform clinical decision-making.An adapted approach was followed by systematically searching online CPG repositories and online databases for LBP CPGs; screening and critically appraising identified CPGs; summarising recommendations from relevant CPGs and organising them into clinical practice activities. Feedback on utility was obtained from 11 physiotherapists.Three high-quality, international CPGs provided 25 recommendations on the assessment and management of acute and subacute LBP relevant to South African physiotherapy practice. They were organised into 10 headings. Physiotherapy user feedback suggested that this document would assist in clinical decision-making.Organised recommendations extracted from multiple, relevant CPGs provide an end-user-friendly resource for physiotherapists treating LBP.Collated and organised CPG recommendations may effectively assist South African physiotherapists’ clinical decision-making in assessing and managing patients with acute and subacute LBP. Clinical practice guidelines (CPGs) are ‘a convenient way of packaging evidence and presenting recommendations to healthcare decision makers’ (p. 6) ’. This essentially refers to Tier 3 documents  as described by Machingaidze et al. :9. Howevde novo development of CPGs using the steps outlined in Guidelines 2.0 may not be practical, feasible or cost-effective. To the authors’ knowledge, there is no South African CPG for the management of acute or subacute LBP. In lieu of writing a de novo CPG for LBP management for South African physiotherapists, which included contextualised Tier 3 documents, we explored how currently available, well-written CPGs might be adapted, adopted or contextualised to guide LBP management for physiotherapists working in South African health care environments , the National Guideline Clearinghouse site and the www.google.com search engine, from inception to January 2019. Nine electronic databases  were also searched from inception to January 2019, to ensure that all eligible CPGs were identified. Search terms and variations thereof used included: ‘clinical practice guidelines AND (acute LBP OR subacute LBP)’. Screening CPGs: Potentially relevant CPGs were screened for scope and purpose relating to physiotherapy assessment and management of adults suffering from acute or subacute LBP. It was essential to determine when selecting CPGs that their purpose clearly linked with the needs of their end-users ; their scope included relevant clinical question(s), and they incorporated the most current and the best available evidence  Checklist was used to critically appraise the methodological quality of potentially relevant CPGs  . All of the clinicians had limited previous exposure to CPG use in practice. The clinicians resided in a rural town, further limiting their exposure to evidence-based practice courses. The acceptability of intervention measure (AIM), intervention appropriateness measure (IAM) and feasibility of intervention measure (FIM) were used to determine whether it would be a useful document in daily clinical practice  as a Tier 3 document to assist clinical decision-making , 514–530, viewed from https://annals.org/aim/fullarticle/2603228/noninvasive.Qaseem, A., Wilt, T.J., McLean, R.M. & Forciea, M.A. 2017, ‘Noninvasive treatments for acute, subacute, and chronic low back pain: A clinical practice guideline from the American College of Physicians’, Evidence-informed primary care management of low back pain: Clinical practice guideline, Toward Optimized Practice, Edmonton, AB, viewed from http://www.topalbertadoctors.org/cpgs/885801.Toward Optimized Practice (TOP) Low Back Pain Working Group, 2017, Low back pain and Sciatica in over 16s: Assessment and management: assessment and non-invasive treatments, National Institute for Health and Care Excellence, viewed from https://www.nice.org.uk/guidance/ng59.National Guideline Centre (Great Britain), 2016, AssessmentAll three CPGs recommend that clinicians should conduct a full assessment prior to devising a management plan. The assessment should include history, assessment for red and yellow flags, and a physical assessment. In addition, two CPGs recommend using a risk stratification tool, such as STarT Back, as part of the full assessment.Red flagsAll three CPGs recommend identifying patients exhibiting red flags (or specific organic causes) for LBP. These include cancer, infection, trauma, inflammatory disease, etc. An important element of the assessment is therefore to determine if patients have recently developed new or changed symptoms that might indicate organic causes.All three CPGs recommend referring a patient with suspected red flags for immediate evaluation and management to an appropriate resource, such as a general practitioner, hospital emergency department or medical specialist, depending on what/who is available in a specific setting and local referral protocols.ImagingAll three CPGs recommend that in the absence of red flags, diagnostic imaging  should not routinely be offered to patients with either acute or subacute LBP.ManagementAll three CPGs recommend advising the patient to stay active and resume normal activity levels as soon as possible.All three CPGs recommend using psychological interventions  to address yellow flags and prevent chronicity after an acute or subacute LBP incident.Two of the CPGs recommend using spinal mobilisation, manipulation or soft tissue techniques (including massage) for managing patients with acute and subacute LBP. However, one of the CPGs reports that there is insufficient evidence to recommend or to advise against spinal mobilisation.One CPG recommends that the clinicians should base their management on risk stratification.Two CPGs advise against offering traction for managing patients with acute and subacute LBP.One CPG advises against offering belts, corsets, foot orthotics or rocker sole shoes to patients with acute and subacute LBP.One CPG advises against prescribing bed rest to patients with acute and subacute LBP.Return to workTwo of the CPGs recommend advising the patient to return to work.Advice and educationAll three CPGs recommend reassurance of the patient about the general favourable prognosis of patients with acute and subacute LBP, regardless of the management.ExerciseTwo of the CPGs recommend advising the patient to start an exercise programme for the management of acute and subacute LBP; however, the type of exercise may vary. Group exercise classes may be of benefit, depending on the patient’s needs and resources available.Electrophysical modalitiesTwo of the CPGs recommend the prescription of superficial heat, cold or alternating heat and cold (for no longer than 15–20 min) for the home management of patients with acute and subacute LBP.Two of the CPGs advise against offering ultrasound for the management of patients with acute and subacute LBP.Two of the CPGs advise against offering transcutaneous electrical nerve stimulation (TENS) for the management of patients with acute and subacute LBP.Two of the CPGs advise against offering interferential therapy for the management of patients with acute and subacute LBP.One CPG advises against offering percutaneous electrical nerve stimulation (PENS) for the management of patients with acute and subacute LBP.There is inconclusive evidence to recommend for or against: acupuncture; back schools; the clinical prediction rule for spinal manipulative therapy; herbal medicine; low-level laser therapy; operant conditioning provided by a physiotherapist; short-wave diathermy and topical NSAIDS®); touch therapies and yoga therapyThere is insufficient evidence to recommend for or against: craniosacral massage/therapy; modified work duties for facilitating return to work; shock-wave treatment; Tapentadol  or anticonvulsants for the management of patients with acute and subacute LBP.CPG, clinical practice guidelines; LBP, low back pain; CT, computed tomography; NSAIDS, non-steroidal anti-inflammatory drugs.Tier 3 utility: The draft Tier 3 document was considered by the participating physiotherapists to be an acceptable, appropriate and feasible end-user document for busy clinicians. This article is one of the few articles that outline the development processes of a Tier 3 document, using recommendations from multiple relevant CPGs for one common condition (LBP). The Tier 3 document complemented a physiotherapy training programme about CPG uptake and thus provides new knowledge to assist in evidence implementation for busy clinicians (Machingaidze et al. The physiotherapists who validated this Tier 3 document believed that it would be a useful quick reference in a busy physiotherapy practice, as it is only two pages in length and is easy to read. Furthermore, they believed that it would be useful to promote evidence-based physiotherapy for LBP to other health care professionals.de novo development of CPGs for busy clinicians in a resource-constrained country, such as South Africa (Grimmer et al. The steps that were followed to develop the Tier 3 document could be used to develop similar summary documents for other conditions, as the physiotherapists indicated that this was the type of document they would find easy to access and apply in practice (Stander et al. The authors acknowledge the limitations of the study. The three parent CPGs did not expand on different approaches to follow with the STarT back risk stratification tool findings (Hill et al. This article presents a step-by-step approach to producing a user-friendly, credible Tier 3 document tailor-made for busy physiotherapy clinicians treating acute and subacute LBP, by using organised recommendations from multiple, relevant CPGs.


Neural codes have been postulated to build efficient representations of the external world. The hippocampus, an encoding system, employs neuronal firing rates and spike phases to encode external space. Although the biophysical origin of such codes is at a single neuronal level, the role of neural components in efficient coding is not understood. The complexity of this problem lies in the dimensionality of the parametric space encompassing neural components, and is amplified by the enormous biological heterogeneity observed in each parameter. A central question that spans encoding systems therefore is how neurons arrive at efficient codes in the face of widespread biological heterogeneities. To answer this, we developed a conductance-based spiking model for phase precession, a phase code of external space exhibited by hippocampal place cells. Our model accounted for several experimental observations on place cell firing and electrophysiology: the emergence of phase precession from exact spike timings of conductance-based models with neuron-specific ion channels and receptors; biological heterogeneities in neural components and excitability; the emergence of subthreshold voltage ramp, increased firing rate, enhanced theta power within the place field; a signature reduction in extracellular theta frequency compared to its intracellular counterpart; and experience-dependent asymmetry in firing-rate profile. We formulated phase-coding efficiency, using Shannon's information theory, as an information maximization problem with spike phase as the response and external space within a single place field as the stimulus. We employed an unbiased stochastic search spanning an 11-dimensional neural space, involving thousands of iterations that accounted for the biophysical richness and neuron-to-neuron heterogeneities. We found a small subset of models that exhibited efficient spatial information transfer through the phase code, and investigated the distinguishing features of this subpopulation at the parametric and functional scales. At the parametric scale, which spans the molecular components that defined the neuron, several nonunique parametric combinations with weak pairwise correlations yielded models with similar high phase-coding efficiency. Importantly, placing additional constraints on these models in terms of matching other aspects of hippocampal neural responses did not hamper parametric degeneracy. We provide quantitative evidence demonstrating this parametric degeneracy to be a consequence of a many-to-one relationship between the different parameters and phase-coding efficiency. At the functional scale, involving the cellular-scale neural properties, our analyses revealed an important higher-order constraint that was exclusive to models exhibiting efficient phase coding. Specifically, we found a counterbalancing negative correlation between neuronal gain and the strength of external synaptic inputs as a critical functional constraint for the emergence of efficient phase coding. These observations implicate intrinsic neural properties as important contributors in effectuating such counterbalance, which can be achieved by recruiting nonunique parametric combinations. Finally, we show that a change in afferent statistics, manifesting as input asymmetry onto these neuronal models, induced an adaptive shift in the phase code that preserved its efficiency. Together, our analyses unveil parametric degeneracy as a mechanism to harness widespread neuron-to-neuron heterogeneity towards accomplishing stable and efficient encoding, provided specific higher-order functional constraints on the relationship of neural gain to external inputs are satisfied. IBiological neural systems encode environmental stimuli in the process of eliciting behavioral responses. From the standpoint of efficient information encoding, these coded representations are required to maximize the information conveyed by the external stimulus. Within an information-theoretic framework, efficient encoding translates to the requirement that the distribution of stimulus attributes match the encoding strategy –12. EffiIn biological systems, such efficient representations emerge despite two critical constraints. The first involves the ubiquitous expression of biological heterogeneities, manifesting as pronounced neuron-to-neuron differences in response properties and underlying molecular components, even between adjacent neurons of the same subtype in the same brain region. These differences imply that the precision in encoding should account for this nonhomogeneous nature of neural parameters and responses. The second constraint corresponds to the limited ranges of the encoding space, placing a requirement that neurons and their circuits employ this range judiciously so as to enhance coding capacity. For instance, if neurons employed their firing rate as the code for a specific attribute of the environmental stimulus, there is a limit on the range of this firing rate based on the cellular and molecular constraints on the neuron. How do biological systems ensure maximal information encoding of environmental stimuli in the face of ubiquitous biological heterogeneity, while constrained by limited coding spaces?within a single place field  matched the electrophysiological values of ~120 MΩ, and the passive charging time constant (=RmCm = 40 ms) was ~40 ms ∞ = 100 nM defines the steady-state value of cytosolic calcium concentration [Ca]i.where α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) was modeled as the sum of currents carried by sodium and potassium ions . We computed the mean and variance of the phases within each stimulus bin si and constructed a normal distribution with these statistics to yield p(ϕ|is) , synaptic strength  mediates phase precession. Our systematic stochastic search demonstrated the requirement of a synergistic balance between intrinsic excitability and synaptic strength in preserving the ability of this input structure in mediating phase precession and in yielding efficient phase coding through disparate routes.Intuitively, the higher frequency of the intracellular theta , in conjunction with the ramp in the voltage driven by afferent synaptic inputs, explain phase precession in this model ,33. SpecDo our conclusions on phase coding depend on the specific choice of the model for phase precession? In the absence of heterogeneous conductance-based implementations of each of these different models, it is infeasible to make predictions on whether our conclusions would depend on the specific model in hand. However, we postulate that our main conclusions should hold irrespective of model choice. First, we postulate the requirement of a balance between synaptic drive and intrinsic excitability as a general principle for achieving efficient phase coding. Second, we hypothesize that ion-channel degeneracy would play a key role in the emergence of concomitant efficient phase codes and signature electrophysiological properties. We base this hypothesis on the several instances across different systems demonstrating ion-channel degeneracy in achieving cellular-and network-scale functions –62,81–85DPmax [There are several competing models on the specific roles of the balance between excitatory and inhibitory synaptic inputs in the emergence of place cells and phase precession ,80,86–92Pmax  could bePmax , and thePmax  would onmodulatory role for intrinsic excitability in the emergence of phase precession, while also ruling out a direct well-defined relationship between intrinsic excitability and phase-coding efficiency [synergistic counterbalance between intrinsic excitability and synaptic strength for the emergence of efficient phase coding systems. Explicitly, as in [mediates phase precession in our model. Here, we demonstrate that neuronal intrinsic excitability and synaptic strength play modulatory roles in preserving the ability of this input structure in mediating phase precession and unveil the requirement for a synergistic counterbalance between them in maintaining high efficiency phase coding. These observations emphasize the need to broaden the scope of the definition of excitation-inhibition (E-I) balance to include neuronal intrinsic excitability (IE) in the assessment of phase coding in particular, and in neuronal and network physiology and pathophysiology in general.An important methodological variation in our study is the systematic incorporation of biological heterogeneities in ion channel conductances and in neuronal intrinsic properties. This is in contrast to models for phase procession, which employ a single hand-tuned model with fixed channel conductances and intrinsic excitability. This methodological variation allowed us to demonstrate a ficiency . Importay, as in , the temy, as in  mediatespassive balance between excitatory and inhibitory synaptic inputs. Neuronal intrinsic properties—including neural excitability, frequency-dependent filtering, spatiotemporal summation, dendritic spike, and plateau potential initiation— could dramatically alter neural function even when identical inputs arrive onto neural structures. Therefore, studies assessing neural coding should assess an active balance that incorporates neuronal intrinsic excitability and ion channel function as a part of this balance. These studies should incorporate the tremendous biological heterogeneities observed in neural excitability, synaptic strength, and ion channel properties, rather than assuming a single model with fixed excitability and/or fixed excitatory/inhibitory synaptic strengths. Together, our study strongly suggests that a passive E-I balance be instead replaced by an active E-I-IE balance in the assessment of neural and network structures under physiological as well as pathophysiological conditions.The discoveries of physiological roles for active dendritic structures in information processing ,93–98, oEWhile emphasizing the phase code from a single neuron perspective, our approach did not account for the rate code or for phase and rate codes from a network perspective where different neurons together encode space by representing different place fields in an arena ,69,111. In the context of experience-dependent asymmetry, our model predicts a temporal adaptation that preserves the efficiency of the phase code when the symmetry of afferent synaptic drive is altered . Our resAccounting for these aspects of experience dependence also requires that the place-cell model accounts for morphological properties of CA1 pyramidal neurons , includispecific structural components expressed in those neuronal subtypes, and should be explored through careful assessment of intrinsic-synaptic counterbalances mediated by ion channels and receptors expressed in those neurons. However, such generalization should specifically account for differences in local/afferent connectivity (including recurrent connections) as well as the specific intrinsic properties and ion channels expressed in these neurons.Our model was limited to phase precession in CA1 pyramidal neurons, focusing on heterogeneities in intrinsic properties owing to baseline variability, activity-dependent plasticity, or neuromodulation. We postulate that our fundamental conclusions would extend to other neuronal subtypes that exhibit phase precession. Specifically, we hypothesize that synergistic interactions between neural gain and external inputs would mediate the expression of degeneracy in the concomitant emergence of efficient phase coding and homeostasis in other neuronal subtypes as well. Such degeneracy would be achieved through disparate combinations of Supplemental Figures S1–S5


Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several malignancies of endothelial and B-cell origin. The fact that latently infected tumor cells in these malignancies do not express classical viral oncogenes suggests that pathogenesis of KSHV-associated disease results from multistep processes that, in addition to constitutive viral gene expression, may require accumulation of cellular alterations. Heritable changes of the epigenome have emerged as an important co-factor that contributes to the pathogenesis of many non-viral cancers. Since KSHV encodes a number of factors that directly or indirectly manipulate host cell chromatin, it is an intriguing possibility that epigenetic reprogramming also contributes to the pathogenesis of KSHV-associated tumors. The fact that heritable histone modifications have also been shown to regulate viral gene expression programs in KSHV-infected tumor cells underlines the importance of epigenetic control during latency and tumorigenesis. We here review what is presently known about the role of epigenetic regulation of viral and host chromatin in KSHV infection and discuss how viral manipulation of these processes may contribute to the development of KSHV-associated disease. According to current estimates, at least 10% of all worldwide cancer cases are attributable to viral infections , 3. The In addition to KS, KSHV is also linked to two B-cell malignancies: primary effusion lymphoma (PEL) and the plasmablastic form of multicentric Castleman disease (MCD) , 12. PELNumerous molecular, immunological, and epidemiological studies have provided overwhelming evidence that KSHV is causally linked to the etiology of the above diseases. However, KSHV’s precise contribution to tumorigenic processes, especially regarding cellular transformation, remains unclear. In contrast to some transforming and/or tumorigenic viruses that encode potent oncogenes  LMP1 protein), no individual KSHV gene product appears to transform primary human cells by itself. Nevertheless, the ability of several viral proteins and non-coding RNAs to inactivate cellular immune or cell cycle check points (reviewed in –25), togGammaherpesvirinae and, together with EBV, forms the human branch of this herpesvirus subfamily. Both EBV and KSHV can cause tumors or hyperproliferative diseases, an ability which they share with several other gammaherpesviruses. Gammaherpesviruses establish long-term reservoirs of infection in lymphocytes . It is thought that these viruses may promote host cell proliferation as a strategy to expand latently infected lymphocyte pools and gain access to long-lived memory lymphocyte compartments. In line with this, gammaherpesviruses have been found to employ sophisticated mechanism to ensure latent genome maintenance in proliferating cells, a feature which is also essential for the maintenance of non-integrated episomes in tumor cells.KSHV is a member of the KSHV has a double-stranded DNA genome of approximately 170 kb. Most of the more than 90 genes encoded by KSHV are required for productive replication. During this phase of the viral life cycle, the so-called lytic genes are transcribed in a cascading manner, resulting in massive amplification of viral genomes, genome packaging, release of new virions, and, ultimately, lysis of the host cell. Like all members of the herpesviruses, KSHV is also able to establish latent infections. Latently infected cells do not produce any viral progeny and remain fully viable, thus allowing the virus to evade host immune surveillance mechanisms and persist for virtually indefinite periods of time. During canonical latency, lytic genes are transcriptionally silenced, and the virus expresses only a minimal set of genes that promote viral persistence and host cell survival. Latent gammaherpesvirus genomes generally persist as non-integrated, fully chromatinized episomes in the nucleus of the infected cells. Since such elements lack a kinetochore, one of the most important functions of latency products is to ensure that viral episomes are properly segregated to daughter cells upon cell division. Indeed, many  gammaherpesviruses encode latently expressed nuclear proteins that tether viral episomes to mitotic host chromosomes, which thus become piggyback vectors for efficient episome partitioning. Additionally, some of these factors have been demonstrated to directly recruit the cellular replication machinery to viral episomes. In KSHV, these functions are mediated by the multifunctional latency-associated nuclear antigen or LANA. LANA binds via a carboxyterminal domain to distinct binding motifs in the viral terminal repeats (TRs), whereas an amino terminal region interacts with histones H1, H2A, and H2B as well as several other chromatin factors –45. As dIn addition to ensuring episome maintenance, KSHV latency products also can promote proliferation and survival of infected host cells. For example, LANA has been reported to interfere with p53, Rb, and β-catenin pathways –48. DuriAll of the above genes are produced from alternatively spliced transcripts that originate from a single promoter upstream of the LANA coding region , 68, 69,These include vIRF3 , a protein with homology to human interferon-regulatory factors (IRFs) that is expressed in latently infected B cells, and vIL-6, a viral homolog of the pro-inflammatory cytokine interleukin 6 (IL-6) which is highly expressed in MCD , 70–72. Given the fact that most proliferating cells in gammaherpesvirus-associated cancers are latently infected, the viral latency program is generally considered the driving force of tumorigenesis. However, despite the many potentially proto-oncogenic functions described above, KSHV latency products alone are unable to robustly transform cells in vitro and also do not efficiently induce tumors in in vivo mouse models. One possible explanation for this fact is that KSHV-encoded latency genes may act in concert with lytic genes to induce tumors. This may seem counterintuitive at first, considering that cells undergoing lytic replication are bound to die. However, several KSHV-encoded factors are secreted from lytic cells or can induce secretion of cellular signaling molecules that can affect other latently infected (or uninfected) cells in a paracrine manner. An important factor among these is vGPCR, a constitutively active G–protein–coupled chemokine receptor homolog  which trThe term “epigenetic” describes heritable phenotypic traits or changes that do not result from alterations of the DNA sequence itself. On the molecular level, most epigenetic phenomena are mediated via regulation of gene accessibility and activity through methylation of DNA  or by post-translational histone modifications (histone PTMs) , we were able to demonstrate that appreciable CpG methylation patterns do not emerge until several weeks following a de novo infection. Instead, viral episomes rapidly and globally acquire H3K27-me3 marks early in infection . The enrAn important feature of H3K27-me3-positive facultative heterochromatin is that it is more dynamic when compared to constitutive heterochromatin. Studies in various organisms have shown that repression by polycomb complexes can be overcome relative easily, and some PRC-bound genes in mammals have been found to frequently switch between silent and transcriptional active states . These oGiven the above, it is likely that KSHV latency represents a rather flexible instead of rigid transcriptional program. This is in line with the observation that the signaling molecules K1 and K15, genes which are highly expressed during the lytic cycle, can also be transcribed at low level in latently infected cell populations , 104. WhHow are PRCs attracted to KSHV episomes and to what extend is this process controlled by the virus? Our own recent data suggest that the composition of the viral genome sequence itself favors rapid silencing by PRCs . There aThe investigation of KSHV-induced cellular epigenome changes, especially in relevant primary cell types, is still a relatively young research field. In large part, this is due to the limited availability of suitable infection systems. Primary lymphocytes are largely refractory to KSHV infection in vitro, a fact that greatly complicates the study of potential B-cell-specific mechanisms. In contrast, primary endothelial cells or mesenchymal stem cells  can be infected in vitro, yet long-term investigation of such cultures is difficult because they are not efficiently immortalized by KSHV and furthermore tends to lose the virus after only a few cell doublings. Most studies have therefore used de novo infection or ectopic expression of viral genes in fully transformed cell lines instead. Hence, a potential caveat is that some of the observations made in these systems could represent artifacts resulting from the use of heterologous cell lines or ectopic overexpression. We will discuss some of the activities which may contribute to the pathogenesis of KSHV-associated disease in the following. In Table Given its constitutive expression and close association with chromatin, LANA is one of the factors which are most likely to influence the cellular epigenome  . LANA-deBesides LANA and vFlip, KSHV-encoded miRNAs have also been found to affect the cellular epigenome Fig. . StrikinIn addition to the above latency genes, several lytic gene products can affect viral and host chromatin  that had been infected with a recombinant KSHV also observed transcriptional changes that correlated with methylation changes in the promoters of several genes . ApproxiAlthough the above findings very strongly suggest that epigenome alterations have a profound effect on viral gene expression and infection-associated tumorigenesis, all hitherto discussed observations have been made in in vitro systems. What is the evidence that such changes also matter in vivo? An interesting study by Naipauer and colleagues  has demoThere is profound evidence that control of latent KSHV infection is intricately linked to epigenetic regulation. Although some of the mechanisms which shape the epigenetic landscape of viral genomes have been discovered, important questions regarding viral epigenome dynamics and adoption of alternate, potentially tumor-promoting latency programs remain. This is particularly true because constitutive latency genes may cooperate with transiently expressed lytic factors to induce stable and heritable changes of the cellular epigenome. Studying the combinatorial effects of such genes, especially in relevant primary cell models, represents a promising direction for future research. In addition to existing in vitro models of endothelial cell infection, newly developed mesenchymal stem cell systems may provide novel opportunities for in vivo studies. Since the majority of PELs also harbors EBV, another interesting yet unexplored aspect is the effect of combined KSHV and EBV gene expression on viral and host epigenomes that can be studied in recently established B-cell models of co-infection . The ava


Efficient communication is essential in all layers of the biological chain. Cells exchange information using a variety of signaling moieties, such as small molecules, proteins, and nucleic acids. Cells carefully package these messages into lipid complexes, collectively named extracellular vesicles (EVs). In this work, we discuss the nature of these cell carriers, categorize them by their origin, explore their role in the homeostasis of healthy tissues, and examine how they regulate the pathophysiology of several diseases. This review will also address the limitations of using EVs for clinical applications and discuss novel methods to engineer nanoparticles to mimic the structure, function, and features of EVs. Using lessons learned from nature and understanding how cells use EVs to communicate across distant sites, we can develop a better understanding of how to tailor the fundamental features of drug delivery carriers to encapsulate various cargos and target specific sites for biomedicine and bioengineering. EVs are nano-sized proteo-lipid vesicles released by cells via exocytosis which are then taken up by recipient cells ,2. They Inspired by the natural efficacy of EVs in cell-cell communication, synthetic nano-based approaches have been developed to mimic and/or amplify the messages delivered by EVs ,25,26. IThere is an assortment of biological messages continuously sent between the various cells of our body to maintain physiological homeostasis. The content of the messages delivered by EVs is affected by the parent cell , environmental cues , and biological parameters . We organized the following sections to describe how different cells use EVs to deliver specific biological messages .The immune system plays a critical role in the maintenance of homeostasis. The body’s immune response to foreign bodies is coordinated by many different cells: macrophages, T-cells, dendritic cells (DCs), and natural killer (NK) cells. These cells are endowed with different objectives and work together to defend our body against infectious organisms and other foreign invaders . EVs relInnate immunity is the body’s first line of defense upon pathogen detection. This arm of the immune system is nonspecific and comprises a general response to kill anything that is not identified as ‘self’. Primary mediators of this response include macrophages, DCs, and NK cells.Salmonella enterica and Mycobaterium tuberculosis carried PAMPs that induced cytokines production via activation of Toll-like receptors (TLRs). Another study demonstrated that exosomes from M. tuberculosis-infected macrophages carry major histocompatibility complex (MHC) class I and II costimulatory molecules capable of inducing memory in CD4+ and CD8+ T-cells.Macrophages are phagocytic cells that continuously circulate throughout the body in search of pathogens. Upon identification of one, macrophages phagocytose bacteria and viruses, process the pathogenic components, produce antigens recognized by effector immune cells, and secrete cytokines as signals to recruit other immune cells to the site of infection. As these cells process the foreign material, they also secrete EVs containing pathogen-associated molecular patterns (PAMPs) . EVs furIn addition to having stimulatory effects on the immune system, macrophage EVs can also possess inhibitory effects. This behavior aligns with the double polarization profile of macrophages as either pro-inflammatory M1 or anti-inflammatory M2. In fact, analysis of miRNA content from activated macrophages has shown that EVs secreted from either M1 or M2 macrophages carry signatures of the corresponding phenotype. As a result, the behavior of these EVs in the disease context mimics that of the source cells. For example, M2-derived EVs promote migration of gastric cancer cells via activation of PI3K-Akt pathway, a behavior that is much like the migratory behavior induced by tumor-associated macrophages (TAMs) across many cancer types. Furthermore, these EVs possessed a molecular profile similar to the pro-tumor, immunosuppressive phenotype observed in TAMs, which includes increased expression of M2 markers (CD163 and CD206) and reduced expression of M1 markers (IRF5 and TNF-a). Additional examples, from a recently published paper by Biemmi et al. , describTherefore, the molecular profile of macrophage-based EVs plays a key role in mediating the immune response within the disease context, where the content of these vesicles is vital to tipping the balance towards a pro- or anti-inflammatory response.DCs are professional antigen-presenting cells (APCs) whose primary function is to capture, process, and present antigens to lymphocytes . This prThe primary role of NK cells in innate immunity is to destroy target cells through the release of cytotoxic molecules or the induction of apoptosis via activation receptor ligation . EVs secThe adaptive immune response can be viewed as a response that is molded and shaped over time as cells are exposed to antigens. This acquired immune response results in greater specificity of the immune response to a particular pathogen. Although components of the innate immune system mediate the activation of this response, the key effector cells involved are T- and B-cells.The ability of T-cells to recognize a specific antigen induces a coordinated two-pronged response. The first involves activation of cytotoxic T-cells to destroy foreign material, while the second involves the priming of memory T-cells to recognize future exposures to the same antigen. T-cell derived EVs contain molecules found in other EV types, such as miRNA, perforins, and granzymes, and express common T-cell markers, including CD3, CD8, and TCR. Stemming from the stimulatory nature of T-cells, EVs secreted by these immune cells have been shown to promote the proliferation of autologous resting cells and promote tumor invasion to the lungs. Activated CD8+ T-cells were also found to inhibit tumor invasion by direct modulation of tumoral MSC via miR-298 . In contKey functions of B-cells include the production of antibodies, the presentation of antigens to T-cells, and the development of immune memory. The release of EVs from B-cells occurs in response to CD40 and IL-4 signaling. Isolated EVs show high expression of B-cell markers , MHC I, MHC II, and tetraspanin proteins (CD81). Investigation of immune cell response to these B-cell derived EVs indicated significant increases in the number of NK, T-, and B-cells within the spleen five days postimmunization. Another study demonstrated that B-cell derived exosomes could not only activate allergen-specific T-cells, but also induced production of the anti-inflammatory cytokines IL-5 and IL-13 . HoweverErythrocytes, ), and platelets are derived from a common progenitor cell. The primary role of RBCs is to transport oxygen from the lungs to the rest of the body and return carbon dioxide from the body back to the lungs for excretion. Platelets primarily serve to initiate the formation of clots in response to injury of blood vessels. In this section, we will focus on the crosstalk and interactions of EVs excreted by RBCs and platelets and the role that these EVs play in regulating homeostasis.RBCs are the most abundant cell that circulates in the blood . Due to Plasmodium falciparum , it was demonstrated that RBCs could release EVs, promote parasite survival in the host, and transmit to other mosquitoes. This EV-based mechanism of communication represents a possible target for the future development of agents capable of blocking the transmission of this parasite. By preventing the release of EVs, it is possible to inhibit the survival of the parasite in the RBCs and prevent transmission to other mosquitos.RBCs-derived EVs were discovered to exert a protective role on RBCs when in the presence of damaging molecules, such as oxidized proteins. This allowed RBCs to remain in circulation rather than being removed during the clearance of dangerous molecules. In addition, after being infected with Platelets are cells that circulate in the blood and play a key role in maintaining homeostasis and blood vessel integrity. When homeostasis is disrupted and bleeding occurs, platelets adhere to the injured blood vessel and initiate the blood coagulation process. Currently, there is a strong interest in the isolation of platelet-derived EVs and their role as functional mediators of the clotting cascade. For example, platelet-derived EVs were found to act as angiogenetic boosters after vascular injury . FurtherStem cells (SCs) are a small population of cells involved in the homeostasis of cells and tissue. Generally speaking, SCs can be divided into two major categories: (1) embryonic stem cells (ESCs) and (2) adult stem cells (ASCs). Both ESCs and ASCs are characterized by unlimited proliferation and self-renewal capabilities. However, while ESCs present pluripotency capacity and can differentiate into the three embryonic germ layers , ASCs can give rise only to the cell subtypes of the specific tissue where they reside. Based on these important differences, the applications for EVs from ESCs and ASCs vary.Due to their unlimited proliferation potential and differentiation ability, ESCs have extensively been explored as a viable tool to repair damaged or diseased tissues. In particular, ESCs have been applied to transplantation, regenerative medicine, myocardial infarction and ischemia , and wouSeveral studies have confirmed the role of EVs in mediating the communication between ESCs and other cell types  ,49,50,51Adult stem cells are a rare cell population in the human body that can repair or replace injured or diseased tissue. Although perceived to have less risk compared to ESCs, ASCs still present technical complications linked to their isolation, culture-induced senescence, immune-mediated reaction, and genetic instability.One of the more studied and applied subsets of ASCs are mesenchymal stem cells (MSCs). MSCs originate from the mesodermal germ layer, but are commonly found in bone marrow, adipose tissue, and the umbilical cord, MSCs are endowed with multipotent potential as they can differentiate, under specific cytokine-mediated stimuli, into osteoblasts, adipocytes, and chondroblasts. In addition, MSCs play a key role in cancer progression and have shown promise in targeting inflammation. On this note, the tumor microenvironment secretes cytokines  that recruit MSCs into the tumor mass. Once in the tumor, MSCs can interact with surrounding cells by (1) modulating gap junctions; (2) enhance the formation of nanotubes ; or (3) indirectly releasing cytokines and EVs . FurtherIn terms of regenerative medicine, MSC-derived EVs have shown promise for the treatment of wounds , tissue During cancer progression, tumor cells acquire traits that allow them to stimulate their own growth, evade apoptosis and the immune system, sustain angiogenesis, invade local tissues, and metastasize to distant organs. Tumor-derived EVs affect several of these processes, conferring to tumor cells aggressive phenotypes , and influencing the tumor microenvironment ,67,68,69The initial idea of using tumor EVs for drug delivery emerged following the discovery of their cell-specific tropism. The tropism of tumor cell EVs is due to the expression of specific surface proteins, such as the tetraspanins, growth factor receptors, and adhesion molecules , which aRather than capitalizing on the specificity of native surface proteins on EVs for targeted delivery, an alternative strategy is to load cells before EV isolation and use the native packaging mechanisms in donor cells to create drug-loaded EVs without post-secretion manipulation. Scientists demonstrated this technique by collecting tumor-derived EVs from various tumor cells  that were initially incubated with chemotherapeutic drugs , and then irradiated with UV light to induce apoptosis and stimulate the formation of drug-loaded EVs . These tIn cancer patients, EVs were found in the spleen and lymph nodes of cancer patients with lymphoma. This led to the discovery that the spontaneous shedding of EVs from highly metastatic cancer cells could transfer their metastatic ability to poorly metastatic cancer cells , which cOther strategies for cancer immunotherapy include activating or stimulating immune responses by activating T-cells to a broad range of tumor antigens. As previously discussed, mature DCs can activate immune responses via T-cells but are limited by the poor antigen uptake and cross-presentation on MHC class I molecules. Interestingly, ascites recovered from cancer patients were discovered to have high levels of tumor-derived EVs endowed with tumor antigens, including MHC I molecules ,84. ThesIn general, drug delivery system developers are seeking ways to optimize nanocarriers’ properties. More specifically, they are trying to improve circulation time, prevent accumulation in filtering organs, and overcome native biological barriers  that prevent specific and directed targeting . While sThe main methods to develop EVs for clinical use include (1) the modification of donor cells before EVs secretion and (2) surface engineering of natural EVs after secretion by donor cells . FollowiNano-engineers have also developed different strategies to alter EVs’ payload after cellular secretion of EVs. This cargo alteration can be done using two different methods: passive and active EVs loading. Passive loading does not involve energy investment or any other external component besides the EVs and the cargo that is to be loaded. Hydrophilic and neutrally charged molecules are able to diffuse through the bilayer membrane based on concentration gradients, while hydrophobic components can integrate within the hydrophobic membrane due to hydrophobic or electrostatic interactions. Zhuang et al. have demonstrated this kind of passive encapsulation using curcumin or JSI124 (cucurbitacin I) into EL-4 exosomes by incubating at 22 °C for 5 min . Active While the engineering of native EVs themselves represents one approach to further modify these vesicles, another strategy involves combining the biological features of EVs with the tunable properties of synthetic NPs. For example, the development of synthetic liposomal nanoparticles (NPs) using either lipids or proteins found in native EVs has been shown to enhance the properties of natural EVs by mimicking their native features ,102. OneNovel engineered EVs with leukocyte membrane proteins were developed in order to combine leukocytes’ biological targeting properties with the nanoparticles’ ability to deliver different types of cargo. The specific biological properties of leukocytes that can be exploited include evasion from the hosts’ immune system, the ability to cross biological barriers, and specific targeting of tissues via their cellular membrane interactions. This allows particles to avoid opsonization from the immune system, reduce sequestration in filtering organs, communicate with endothelial cells through receptor–ligand interactions, and transport a payload across an inflamed reconstructed endothelium ,108,109.3O4) NPs. ‘NKsomes’, which consisted of doxorubicin-loaded NK cell membrane coated nanoparticles, exhibiting superior affinity to tumor cells while maintaining a circulation time of 18h in the blood [In addition to the use of whole leukocyte populations for novel engineered EVs, researchers have explored the use of specific immune cell membranes for a variety of applications. Given their key roles in mediating and executing the immune response in different disease conditions, T-cells and macrophages have been commonly used immune cell sources for these EVs. For example, CD4+ T-cell membrane coated polymeric nanoparticles were developed to target HIV viral particles and were shown to act as decoys, preventing the virus from attacking the intended host targets. In the context of sepsis, macrophage-coated nanoparticles have been utilized for endotoxin neutralization and the sequestration of inflammatory genes. These engineered EVs demonstrated their ability to directly modulate the immune response involved with the disease. In the context of cancer, immune-cell-based engineered EVs have been used as both drug delivery systems and agents for photothermal therapy. More specifically, macrophage membrane-derived vesicles were coated onto Magnetite  . CriticaNovel engineered EVs with membrane proteins from stem cells were developed in order to regulate the inflammatory response and to explore their potential use as an anti-tumor therapy. The majority of attempts to engineer these stem cell EVs have been done in vitro by inducing genetic modifications on the parent cells . These modifications produced stem cell EVs that demonstrated superior therapeutic efficacy.MSCs can be engineered in vitro by stimulation with different cytokines  in order to increase the expression of programmed death-ligand 1 (PDL-1) and MHC class II molecules on secreted EVs. These secreted EVs can then be used for applications where regulation of the inflammatory response is needed . For exaThe natural tropism of cancer cell-derived EVs for tumors, the pre-metastatic niche, and their potential use for immunotherapy inspired researchers to create engineered tumor EVs. As a proof-of-concept, researchers demonstrated that engineered EVs created by coating PLGA nanoparticles with membranes from MDA-MB-435 and B16-F10 cells exhibited a successful in vitro immune response in T-cells and targeting of cancer cells. For immunotherapy, researchers used engineered EVs from B16-F10 incorporated with monophosphosphoryl lipid A and showed promising in vitro results with a significant upregulation of DCs maturation markers that led to the stimulation of T-cells. For drug delivery applications, engineered cancer EVs took advantage of the inherent homotypic binding observed in cancer cells . EngineeThe possibility of developing EVs that have the ability to target cancer cells spurred the development of additional engineered cancer EVs for cancer therapy. Different cancer EVs were created by following a similar top-down approach to coat nanoparticles with cancer cell membranes. By taking advantage of the homotypic binding of cancer cells, investigators tested the ability of 4T1 (murine breast cancer cells) to engineer cancer-engineered EVs for in vivo therapy ,122. In Effective tumor therapy was also achieved using engineered cancer EVs that were loaded with chemotherapy in order to overcome the drug resistance of tumor repopulating cells . Using tEngineered cancer EVs were also created to elicit antitumor immunity for vaccine development. These EVs were composed of PLGA nanoparticles loaded with CpG oligodeoxynucleotide 1826, a potent immunological adjuvant, and coated with cell membranes from murine B16-F10 melanoma cells. These engineered melanoma cancer cell EVs exhibited potent DCs maturation in vitro and in vivo that resulted in a significant generation of native T cells. Furthermore, these engineered cancer EVs demonstrated effectiveness in the prophylactic setting. Vaccination with engineered cancer EVs showed that 86% of mice had no tumor occurrence 150 days after tumor cell challenge, whereas controls had a median survival of only 20 days. However, the investigators discovered that engineered cancer EVs alone were not sufficient for treating tumor cells previously implanted. To achieve adequate efficacy, it was determined that combination therapy of engineered cancer EVs with a cocktail of checkpoint inhibitors was required. Using this combination strategy, engineered cancer EVs plus a checkpoint inhibitor, provided a significant survival advantage, extending the median survival by 11 days compared to either engineered cancer EVs or the checkpoint inhibitor cocktail alone. This demonstrated that engineered cancer EVs were able to act synergistically with anticancer therapies, hyperthermia, photodynamic therapy, and immunotherapies to modulate the tumor microenvironment and immunity to provide elegant solutions for cancer therapy, imaging, and vaccine development.In this review, we highlighted the biological importance of paracrine cell-to-cell communication and elaborated on how different cells can send specific messages to communicate and regulate physiological of pathological events. In addition, we described different methods to engineer EVs to enhance their performance and use them for therapeutic applications.Although EV-based treatments hold great therapeutic potential, several substantial hurdles remain to be addressed before utilizing EVs in clinical practice. Standard operating procedures (SOPs) for their isolation , charactTo date, several standard procedures have been utilized for the isolation and characterization of EVs. Researchers have divided the isolation techniques for EVs into three main classes: ultracentrifugation, adsorption to magnetic/non-magnetic beads, and size exclusion chromatography . In addiAdequate storage methods for EVs have also not been established. Nevertheless, storage methods are urgently needed for the clinical use of EVs as some conditions can alter both the physical and biological properties of EVs. For example, some studies reported that reducing the temperature for antibacterial purposes can directly affect the size and the number of EVs, while the addition of cryoprotectants will result in the partial lysis of EVs ,130. TheThere are many open questions and challenges that need to be addressed before any substantial progress can be made in order to use EVs in clinical practice. Ongoing research has focused on developing the best EV platform for the target application by mimicking either the EV’s surface or morphology. To evade the immune system, leukocytes-mimetic have been developed by integrating leukocytes membrane protein into the EVs’ lipid bilayer. To achieve higher circulation time, RBCs and platelet EVs have been fabricated by either (a) engineering the EVs’ surface with RBCs and platelet membrane proteins or (b) by mimicking the unique morphology of either of these cells. MSCs EVs have been selected to decrease inflammation and tissue regeneration. Notably, these EV surfaces were not only engineered by integrating MSCs membrane proteins but also their membrane lipids. Cancer cell EVs have been used in order to specifically target cancer infected cells by integrating the cancer cells’ membrane proteins to the EVs’ surface. There is no rule of thumb to define EVs that best mimics the true biological state. Therefore, each engineered EV should be designed for its future biological task. Fabrication challenges include: (1) the amount of proteins on each EV membrane; (2) the ratio between the proteins to the EV backbone  on each EV; and (3) the number of injected EVs. All these properties should be taken into consideration when designing EVs. Moreover, scientists must determine how to best define and measure the physiological properties of EVs. By defining these properties, it would then be possible to determine which EVs are ready to be translated for efficient therapy for humans. Finally, in order for EVs to truly be used in patients, large-scale manufacturing procedures that do not interfere with the therapeutic potential of EVs must be developed. Without the ability to scale-up the engineering of EVs, it will be prohibitively expensive to use them in clinical practice.Overall, while nanomedicine has showcased substantial promise in the delivery of therapeutics, a new generation of delivery systems is emerging stemming from biomimetic-based approaches. Using the body’s own tools to communicate, coupled with engineered upgrades , EVs have the potential to provide significant advantages over current synthetic drug delivery systems. These systems will help overcome major challenges faced by native EVs, including scalability, reproducibility, and limited cargo encapsulation. Although each biomimetic EV will demand additional studies to address stability, toxicity, biodistribution, pharmacokinetics, and efficacy, these engineered systems have been shown to mimic native EVs by transferring specific biological markers to synthetic delivery systems or by mimicking natural EVs in shape and size. Utilizing the synergistic effect of the engineered systems to convey specific biological messages holds tremendous promise and potential to improve future therapeutic outcomes.Future studies should focus on EVs engineering and will require the complex molecular composition of these structures for specific therapeutic applications. However, such strategies require the development of novel biotechnological techniques allowing for the precise tuning and characterization of EVs to specific cell types or tissues. In addition, determining what cargo needs to be loaded into EVs to optimize specific and direct biological pathways is necessary. To advance the current engineering of EVs, new techniques are urgently needed that remove unnecessary components and, at the same time, isolate specific proteins. With these tools in hand, EVs can be manufactured without compromising their overall function.Nevertheless, considerable work is currently ongoing to decipher cell–cell communication in an effort to develop novel therapies for a variety of diseases. The unique biological message encrypted within the EV’s “cloak”  could hold the promise of gaining insight into how to better recognize, interact, communicate, and regulate various cellular functions. By transferring these properties to engineered EVs, we could achieve superior success by utilizing and capitalizing on the body’s natural properties.This work has categorized EVs by the origin of specific cell carriers, described their physiological role, and summarized the updated literature surrounding this topic. It also discussed the main challenges and roadblocks that prevent EVs from being used in clinical practice. Finally, this paper reviews novel methods to synthetically fabricate EVs while still emphasizing the importance of mimicking the native properties and functions of cells to best exploit their use for biomedicine and bioengineering.


Extracellular vesicles (EVs) are small membranous particles that can mediate cell-to-cellcommunication and which are divided into at least three categories according to theirsubcellular origin and size: exosomes, microvesicles, and apoptotic bodies. Exosomes arethe smallest (30–150 nm) of these EVs, and play an important role in EV-mediatedcell-to-cell interactions, by transferring proteins, nucleic acids and, lipids from theirparental cells to adjacent or distant cells to alter their phenotypes. Most exosomestudies in the past two decades have focused on their nucleic acid composition and theirtransfer of mRNAs and microRNAs to neighboring cells. However, exosomes also carryspecific membrane proteins that can identify the physiological and pathological states oftheir parental cells or indicate their preferential target cells or tissues. Exosomemembrane protein expression can also be directly employed or modified to allow exosomes toserve as drug delivery systems and therapeutic platforms, including in targeted therapyapproaches. This review will briefly summarize information on exosome membrane proteinscomponents and their role in exosome–cell interactions, including proteins associated withspecific cell-interactions and diseases, and the potential for using exosome membraneproteins in therapeutic targeting approaches. Selective surface modification of exosomes with targetingproteins or peptides and/or therapeutic drugs is a major strategy for exosome-basedtherapeutics.This review focuses on the role of membrane proteins in potential exosome therapeutics, andwill discuss select exosome membrane proteins and their role in exosome-mediated cellcommunication, including proteins associated with cell- or tissue-specific exosomeinteractions, changes in these protein under pathological conditions, and the application ofspecific membrane proteins in disease diagnosis and treatment. Finally, this review willdiscuss the potential advantages and disadvantages of applications that employ, modify, ormimic exosome properties for clinical therapeutics.Exosomes can carry a multitude of diverse factors, and more research is required toevaluate what roles specific factors play in different physiological and pathologicalprocess. Exosomes contain an array of membrane-associated lipids and proteins in addition totheir lipid, protein coding and non-coding RNA cargoes.Many exosome-specific or -selective proteins are displayed on exosome membranes, includingseveral tetraspanin proteins  and proteins involved in celladhesion and signaling, cytoskeletal structures, lipid rafts, and membrane trafficking.http://www.exocarta.org) listing 41 860 exosome-associated proteins identifiedamong 10 analyzed species.Despite the prevalence of a select set of proteins on exosomes derived from diverse celltypes, exosomes can exhibit a large array of proteins depending on their parental cell type,with the Exocarta database .,in vivo therapeutic interventions, but it may be possible toinhibit interactions between specific exosome and cell populations by blocking receptorsinvolved in these selective events.As endocytosis appears to be primarily responsible for exosome uptake, which is requiredfor most exosome-mediated effects to alter the phenotype of their recipient cells, severalapproaches using well-known inhibitors of endocytosis have been examined for their abilityto block exosome uptake and their regulatory effects, including shRNA transfection, lossof function mutations, and small molecular inhibitors, such as genistein andnystatin.While endocytosis appears to represent the dominant means by which exosomes interact withand influence the phenotype of their recipient cells, exosome-associated proteins havealso been implicated in directly regulating the fusion of the exosome and plasmamembranes.,Receptor-mediated membrane fusion has also been implicated in another reproductiveprocess, the interaction of eggs and sperm. EVs are released from the perivitelline spaceof mammalian eggs just prior to fertilization in a mechanism that appears to facilitatefusion of the sperm and egg cell membranes via a process that requires expression ofIZUMO1 by sperm and CD9 by the egg.Exosome membrane compositions can vary based on cell origin, as well as the physiologicalstate of the parent cell during exosome biogenesis, including changes associated withchronic and infectious diseases, suggesting that analysis of specific circulating exosomepopulations could provide valuable information about the physiologic state of tissues thatwould be otherwise difficult to evaluate because of their inaccessibility or the need tosample multiple discrete sites.As surface-bound proteins on exosomes are influenced by the repertoire of proteinsexpressed on the plasma membranes of their parental cells, exosome membrane compositionscan vary based on both the type and the physiological state of their parental cells.The circulating exosome population is highly diverse, as it reflects the aggregatecontributions of all cells and tissues collected during normal systemic perfusion. Theability to detect, isolate, and analyze tissue-specific or cell-specific exosomes in thismixed population could provide valuable information about the physiologic state of tissuesthat would be otherwise difficult to evaluate because of their inaccessibility or the needto sample multiple discrete sites. The potential of such approaches is limited by the lackof validated cell-specific biomarkers, in part resulting from the technical challengesassociated with identifying such markers.Studies designed to analyze cell-specific exosome biomarkers typically employ proteomicsto analyze the differential composition of exosomes derived from distinct cell or tissuesources, and results can be influenced by the state of the cells or tissue analyzed in thestudy and the purity of the source cells and resulting exosome preparations. Research isongoing to identify and validate markers that can distinguish exosomes derived frominfected cells or that can distinguish or target specific cell types, because of the greatpotential for diagnostic and therapeutic applications that could be developed onvalidation of such markers. For example, one recent proteomics study performed withexosomes isolated from primary rat hepatocyte cultures has proposed that the transmembraneprotein ASGR represents a specific marker for hepatocyte-derived exosomes,+ T cell responses,,,Antigen-presenting cells (APCs), including dendritic cells (DCs), macrophages and Bcells, secrete exosomes that tend to display a surface pattern of immune regulatoryproteins and antigens similar to that of their parental cells. Exosome display or deliveryof these proteins can exert the same effects as their expression on their parent cells,stimulating CD4M. tuberculosis culture filtrateprotein, have been employed to develop an exosome-based vaccine which has the ability toactivate an immune response to this pathogen.These proteins can be directly exploited for exosome therapeutics. For example, exosomesisolated from macrophages treated with Exosomes produced by cells experiencing pathological conditions or other stresses canexhibit altered compositions to serve as potential biomarkers of these states. Specificstressors known to alter exosome composition include specific cellular dysfunctions,cancers, and infections.,,,,,,Exosome expression of pathogen-derived factors represents a clear case where thedetection of a target biomarker on a circulating exosome population represents strongevidence of the linked disease. Exosome-derived biomarkers for chronic non-infectiousdiseases and conditions are more challenging to use as they often reflect alteredexpression of a protein that may already be expressed at significant levels in the generalexosome populations, and thus may require target exosomes to be isolated for analysis orthat a threshold be employed to discriminate expression levels characteristic ofpathologic versus non-pathologic conditions.,,,Exosome membrane compositions can change during disease progression, and may thus beuseful as diagnostic or predictive biomarkers of the current disease stage and thepotential for progression to more severe or advanced disease. Exosomes derived frommetastatic cells are reported to carry factors that promote cancer cell proliferation,migration, invasion and angiogenesis, while exosomes from non-metastatic cells tend tocontain proteins involved in cell–cell or cell–matrix adhesion and polaritymaintenance.Circulating levels of cell- or tissue-restricted exosome proteins  can also,clinicaltrials.gov when “exosome” wasused as a search term, indicating the growing interest in exosomes for clinicalapplications. The sections below briefly summarize the major exosome-related therapeuticapproaches under development.Exosomes have multiple advantages for the delivery of therapeutic cargos.,,Exosome membranes are enriched in tetraspanin and heat shock proteins when compared tothose of their parental cells, and these factors and other factors present on membranes ofexosomes derived from healthy cells can exert beneficial physiologic effects on theirrecipient cells.,Exosome proteins that exhibit broad and restricted expression profiles have both beenreported to exhibit specific therapeutic potential. For example, in the former group,exosome CD9 expression has been linked to osteoclastogenesis that can promote osteoblastfusion and bone healing,Most clinically approved drugs are rapidly cleared from the circulation andnon-specifically distributed through body tissues so that only a small fraction of theadministered dose reaches its intended target site, leading to low efficacy and adverseside effects.,Several approaches can be employed to directly modify the surface of native exosomes todisplay ligands or receptors and therapeutic agents facilitate efficient highconcentration delivery of therapeutic drugs to specific target sites. Relatively simplechemical and physical methods can be employed to add therapeutic drugs to the exosomesurface, or its cargo, to expand the range of functional targets and applications that canbe addressed by exosome membrane-based therapy. One approach is to use a freeze–thawprocess to generate exosome–liposome hybrids that exhibit the targeting properties ofexosomes and the surface and cargo modification potential of liposomes, to transfer bothhydrophobic and hydrophilic therapeutic agents.Mixing exosomes directly with candidate proteins to modify their specificity or alter theability to induce functional changes in their recipient cells has not proven successful,however, as this approach has resulted in large, unstable and/or aggregated exosomes, aswell as undesired chemical contamination. Such surface display approaches are alsovulnerable to microenvironment changes encountered during administration that could causeadhered proteins to dissociate, resulting in loss of the target specificity orfunctionality. Exosomes modified by such surface display approaches have not been used forclinical applications because of concerns about their stability and safety.Non–covalent exosome modification approaches have yet to prove viable for exosometargeting or therapeutic delivery, but chemical conjugation has been successfully employedto confer target specificity on native exosomes. One such approach used a reactivecrosslinker approach to attach reactive dibenzolcyclooctyne groups onto amine-containingmolecules on native exosomes, and subsequently reacted these groups with a cyclo(Arg-Gly-Asp-D-Tyr-Lys) [c(RGDyK)] peptide that exhibits specificity for integrin αvβ3,which is expressed on cerebral vascular endothelial in response to ischemia.Such direct modification approaches to alter exosome specificity have the advantage thatthey do not require the identification and rigorous purification of exosomes with adesired target specificity. However, covalent exosome modification approaches carry therisk of undesirable chemical contamination or modification of the altered targetingexosomes, while non-covalent modification approaches may produce exosomes with unstabletarget specificity under physiological conditions. The efficiency of exosome modificationmay also be an issue for both covalent and noncovalent approaches, resulting in exosomepopulations with variable purity and raising the potential for off-target effects.Recombinant DNA approaches have also been employed to generate cell lines that secreteexosomes with desired target specificities, avoiding potential chemical toxicity andreceptor instability problems possible with chemically or non-covalently modifiedexosomes, while ensuring that all exosomes have the desired target specificity. Severalgroups have used recombinant DNA approaches to produce exosomes with engineeredtherapeutic or targeting potentials. For example, genetic engineering has been used tomodify HEK293T cells to generate exosomes that overexpress SIRPα to act as a cancertherapeutic by disrupting CD47-SIRPα interactions between tumor cells and macrophages toattenuate the ability of tumor cells to resist phagocytosis.Modifying exosome membrane expression by genetic alterations to parental cells shouldproduce more uniform exosome populations, with potentially more stable targetingspecificities, than other methods. However, genetic modification approaches are also moretime-consuming and expensive than direct modification approaches to alter targetspecificity, and can raise potential safety concerns that may complicate translation toclinical applications.Neither the surface display nor the genetic engineering approaches described aboveaddress the issue of exosome purification, which can require large sample volumes toobtain sufficient material for therapeutic applications. Exosome mimetics, nanoparticlescoated with membranes that simulate those of exosomes with desired targeting or functionalcharacteristics, have become a promising means to address low yields associated withpurification of endogenous endosomes for therapeutic applications. Exosome mimetics can berapidly and cost-effectively engineered to carry the functional and targeting propertiesof exosomes coupled with the drug loading properties of their nanoparticle cores, and haverecently received considerable attention as effective drug delivery platforms.Membrane-coated nanoparticles generated by extruding biocompatible nanoparticles through ananoporous membrane in the presence of membrane isolates from cells with desiredfunctional properties reveal size distribution, morphology, stability andimmunocompatibility characteristics similar to the characteristics of exosomes produced bythese cells.Tumor-derived exosome membranes can carry both tumor antigens and exhibit specificcell-homing properties. Nanoparticles coated with cancer cell membranes (CCNPs) canpromote tumor-specific immune responses, and have been used as cancer vaccines inconjunction with an immunological adjuvant.Coating polymeric nanoparticles with cell membrane material represents an effectivemethod for introducing multiple desirable membrane antigens and surface functionalities,which is difficult to achieve using traditional synthetic techniques. Preparation of suchexosome-mimetics requires expertise with the isolation of cell membranes and particlefunctionalization, as well as special synthetic equipment, but coating polymericnanoparticles with CCNPs presents an effective means to generate therapeutic agents forcancer immunotherapy and drug delivery, which can be customized for personalized cancertherapy.,,de novo membrane protein or peptideexpression to alter the target specificity or functional activity of the engineeredexosomes. This approach has been employed to produce exosomes that express SIRPα and blockCD47-SIRPα interactions between tumor cells and T lymphocytes,,Native or surface-modified exosomes, exosomes produced by genetically engineered cells,or exosome mimetics can be used for the targeted transport of drugs to diseased cells invarious applications, each of which has its own advantages and disadvantages, assummarized in All cells appear to secrete exosomes, and exosomes are present in all body fluids. Exosomescarry biological and genetic information that can identify their parental cell types, andcan transfer their native or engineered contents to specific recipient cell types throughvarious interaction mechanisms to influence their phenotypes and fates. These propertieshave led to research on the development of exosome biomarkers of disease and therapeuticstrategies that employ the properties of native or modified exosomes or exosome mimetics.Most current exosome biomarker research focuses on serum/plasma exosome analysis forcorrelations with tumor phenotypes, but there is also potential for analysis of exosomes inother tissues to advance the diagnosis of specific tumor types .Proteomics and living cell imaging have led to the discovery that exosome membrane proteinsplay key roles in exosome-mediated information transfer. Characterization ofdisease-specific exosome membrane proteins and better understanding of the physio-pathologicroles of these proteins in their respective diseases has significant implications for thedevelopment of future clinical applications using this information for improved diagnosticsor therapeutics.Exosomes have advantages over many other therapeutic platforms  for the delivery of membraneproteins or drug cargoes. However, low yield rates of both native and modified or engineeredexosomes, and concern over the safety and off-target effects of their additional componentsserve to limit their path to clinical applications. Direct or indirect modification ofexosome membrane proteins can expand the function and application scope of exosometherapeutics, but further increase concerns about the purification, characterization, andsafety of the resulting exosome therapeutics. Recent research has appeared to focus on thedevelopment of nanoparticle-based exosome mimetics that can be precisely engineered todisplay desired characteristics; however, additional validation studies are required todemonstrate that exosome-based therapeutics are suitable for clinical applications.Q.H and H.S contribute equally to this work. Conceptualization, M.H.W. and T.Y.H.;writing—original draft preparation, Q.H; writing—review and editing, C.L. and W.F.T.;visualization, H.S. and J.L.; supervision, W.F.T. and T.Y.H.; All the authors read thearticle and approved the final version.


Despite recent improvements in the burden of cardiovascular disease (CVD) in the UK, deaths from CVD are relatively high compared with other high-income countries. An estimated 7 million people in the UK are living with CVD, and the healthcare cost is approximately £11 billion annually. In more than 90% of cases, the risk of a first heart attack is thought to be related to modifiable risk factors including smoking, poor diet, lipidemia, high blood pressure, inactivity, obesity and excess alcohol consumption. The aim of the study is to synthesise evidence for the comparative effectiveness and cost-effectiveness of different interventions for the primary prevention of CVD.We will systematically search databases , Embase (Ovid), Cochrane Library) and the reference lists of previous systematic reviews for randomised controlled trials that assess the effectiveness and cost-effectiveness of any form of intervention aimed at adult populations for the primary prevention of CVD, including but not limited to lipid lowering medications, blood pressure lowering medications, antiplatelet agents, nutritional supplements, dietary interventions, health promotion programmes, physical activity interventions or structural and policy interventions. Interventions may or may not be targeted at high-risk groups. Publications from any year will be considered for inclusion. The primary outcome will be all cause mortality. Secondary outcomes will be cardiovascular diseases related mortality, major cardiovascular events, coronary heart disease, incremental costs per quality-adjusted life years gained. If data permits, we will use network meta-analysis to compare and rank effectiveness of different interventions, and test effect modification of intervention effectiveness using subgroup analyses and meta-regression analyses.The results will be important for policymakers when making decisions between multiple possible alternative strategies to prevent CVD. Compared to results from existing multiple separate pairwise meta-analyses, this overarching synthesis of all relevant work will enhance decision-making. The findings will be crucial to inform evidence-based priorities and guidelines for policies and planning prevention strategies of CVD.CRD42019123940.PROSPERO  Cardiovascular disease (CVD) includes all the diseases of the heart and circulation including coronary heart disease (CHD) and stroke. CVD accounts for the highest proportion of non-communicable disease deaths, resulting in 160,000 deaths in the UK annually . CardiovThough there are many pairwise systematic reviews and meta-analyses that have examined the effectiveness of drug, lifestyle and policy/structural interventions either separately and collectively  and is reported in accordance with the Preferred Reporting Items for Systematic review and Meta-Analysis Protocols (PRISMA-P) checklist , and theWe will evaluate each identified study against the following selection criteria:Study population: adult populations (≥18 years of age) included in population-based studies, which may or may not be targeted at moderate/high CVD risk groups . As the review focuses on the primary prevention of CVD, we will exclude trials that included those who have experienced a previous myocardial infarction (MI), stroke, revascularisation procedure (coronary artery bypass grafting (CABG) or per cutaneous transluminal coronary angioplasty (PTCA)), and those with angina or angiographically defined coronary heart disease (CHD). Studies with mixed populations, i.e. both population with and without CVD will be included if data for the relevant primary prevention can be extracted.Intervention: any form of intervention aimed at the primary prevention of CVD, including but not limited to drugs , diet , physical activity or public health  Table .Table 1Comparators: other forms of intervention , placebo, usual care or no intervention control group or wait list control.Outcome measures: The primary outcome will be all cause mortality. Secondary outcomes will be cardiovascular diseases related mortality, major cardiovascular events , coronary heart disease , incremental costs per quality-adjusted life years gained reported alongside a randomised trial.Study design: randomised controlled trials (RCTs) of at least 6 months’ duration of follow-up. Units of randomisation could be either individuals or clusters .Due to the likelihood of a high volume of relevant trials to be included, we will follow standard guidelines for integrating existing systematic reviews into new reviews , 9. Whera) A comprehensive literature search for existing systematic reviews will be developed iteratively and undertaken in major medical and health-related electronic bibliographic databases including MEDLINE (Ovid), Embase (Ovid), Cochrane Database of Systematic Reviews (Wiley) and DARE (CRD). Systematic reviews that potentially include primary studies meeting our inclusion criteria will be selected. Records for the included  studies in all selected systematic reviews will be identified and imported into EndNote using HubMed Citation Finder , systemaThe most recent systematic review for any intervention or intervention category will be assessed using AMSTAR-2  items 4,.b) The search for recent trials will be developed iteratively and will be informed by records of a broad cross-section of known studies. Searching based on the concepts of prevention and CVD outcomes, or on intervention terms and CVD outcomes, will be considered and tested. Scoping searches have retrieved very high numbers of trials in Cochrane Controlled Register of Trials (CENTRAL) (Wiley) using either approach. Our trial search will focus on CENTRAL (Wiley). For any interventions where no acceptable systematic review is available, a search for trials with no date limit and including relevant intervention terms will be performed. A draft search strategy in CENTRAL (Wiley) is available in Additional file 3c) In order to capture studies with more obscure records and those that may have been excluded by previous systematic reviews, we will run a highly sensitive search with no date limit and use machine learning to identify a proportion of these records for screening (see ‘Selection Process’ below).d) Finally, the reference lists of included studies will be examined for additional relevant studies.We will develop searches iteratively, referring to known articles, existing strategies and assessed search filters . Search In order to reduce the workload of screening the searches result from the highly sensitive search with no date limit, we will develop a bespoke classifier/algorithm to identify potentially relevant studies. We will aim to achieve a high-performing algorithm comparable to human screening . The comData will be independently extracted using a pre-specified piloted proforma by two reviewers, with discrepancies resolved by a third reviewer. We will use a data collection form for study characteristics and outcome data. One author will extract study characteristics from the included studies and a second author will spot-checked study characteristics for accuracy against the trial report. Any inconsistencies will be resolved by discussion.We will extract the following characteristics:Study citation: year(s) of study, registration number to trial registries, year of publication, location, setting, number of centres, sample size, diagnostic criteria, funding/sponsorMethods: including study design (type of RCT), number of arms, risk of bias (see below)Participants: number, mean age, age range, gender, severity of condition, diagnostic criteria, baseline measures of physiological functioning , inclusion and exclusion criteriaInterventions: intervention, comparison, concomitant medications and excluded medicationsOutcomes: primary and secondary outcomes specified and collected, and time points reported including information on whether an intention to treat approach has been used and how it was definedWe will report dichotomous outcomes as risk ratios (RRs). For continuous outcomes, we will calculate mean differences (MDs) when the studies report homogenous outcomes. Time-to-event outcomes or generic inverse variance outcomes will be expressed as the logarithm of hazard ratio (HR).r = 0.5 between baseline and follow-up to estimate SD for change from baseline. Using the imputed correlation coefficient values, we will calculate SDs for the change from baseline for the studies with missing SDs using the following formula:If possible, we will use the intention-to-treat population for all analyses. When effect sizes are incompletely reported, we will contact the corresponding author. When the SDs of absolute changes from baseline are not available from individual trials, we will impute them as described in detail in the Cochrane Handbook . In brieWe will include cluster-randomised trials in the meta-analysis along with individually randomised trials . Cluster-randomised trials will be labelled with a (C). For cluster-randomised trials to be included in the network meta-analyses, we will adjust for design effect using an ‘approximation method’  if the tFor each identified study that meets the selection criteria, we will extract the following data: country, study design, population, intervention(s), comparator(s), type of economic analysis, perspective, model type (structure and key assumptions), time horizon, effectiveness data, primary outcome, resource use and unit cost data, price year, discounting and the results of the base-case and sensitivity analyses. Data such as outcomes and characteristics will be synthesised quantitatively, where appropriate or narratively. For the primary outcome, the preferred measure will be cost per quality-adjusted life years (QALY) gained.A network plot will be created to describe and present the geometry of the intervention network of comparisons across trials . InterveWe will use the Cochrane Collaboration’s tool for assessing risk of bias for quality assessment of the included trials at trial level . The triFor cluster-randomised trials, we will assess the following cluster-specific risks of bias as outlined in the Cochrane Handbook for Systematic Reviews of Interventions .Recruitment bias - whether the individuals participating in the trial were blinded to the type of cluster they were in before agreeing to participateBaseline imbalance - whether there were differences in baseline characteristics between the randomised groupsLoss of clusters - whether any complete clusters were lost to follow-up and the reasonsIncorrect analysis - whether the proper statistical analysis was carried out for a cluster-randomised designComparability with individually randomised trials - whether the cluster-randomisation method could have resulted in different intervention effects than an individually randomised trialWhen considering treatment effects, we will take into account the risk of bias of the studies that contribute to that outcome.We will produce descriptive statistics and study population characteristics across all eligible trials, describing the types of comparisons and other clinical or methodological variables, such as age, co-medication, country and study follow-up period.In the first step we will perform series of conventional pairwise random-effects meta-analyses by combining studies that compared the same interventions, including the comparison between active treatments and the different control arms. If very few trials are available or the requirements of network meta-analysis are not met, it can be that network meta-analysis will not be appropriate and, in this case, conventional pairwise meta-analysis will be applied.We will assess between-trials heterogeneity (variability in relative treatment effects within the same treatment comparison) using the tau-squared (the variance of the random effects distribution). The heterogeneity variance will be assumed common across the various treatment comparisons (grouped by comparison type) and we will compare the empirical distribution with predictive distributions –23. We wThe transitivity assumption will be assessed by investigating the distribution of clinical and methodological variables that can act as effect modifiers across treatment comparisons .We will assume that patients who fulfil the inclusion criteria are equally likely to be randomised to any of the interventions of interest (i.e. jointly randomisable). When additional evidence of intransitivity is lacking and potential effect modifiers have similar distributions across the included studies, network meta-analysis is likely to give valid results.We will conduct network meta-analyses , 26 to cWe will calculate the probability of a given intervention having the largest beneficial effects as the proportion of simulations in which that intervention will be ranked as the ‘best’ according to the relative prevention effect estimate. In addition, we will calculate alternative rankings  because in some policy and practice areas, the best intervention might be unavailable, too costly or contraindicated. Probability values will be summarised and reported as surface under the cumulative ranking (SUCRA) and graphically ranked using rankograms. SUCRA = 1 if an intervention always ranks first and SUCRA = 0 if it always ranks last.All the network meta-analyses analyses , 26 willThe evaluation of transitivity will be supplemented with a statistical evaluation of consistency, the agreement between direct and indirect evidence. We will evaluate consistencies between direct and indirect comparisons in the network of evidence using the method of ‘node-splitting’ , by calcWe anticipate several sources of heterogeneity relating to the content of the intervention and study design. We will test effect modification of intervention effectiveness using subgroup analyses and meta-regression analyses. For example, where there are sufficient data, we will stratify our analyses (subgroup) by the following: population risk groups , trial period (older versus recent), sex  and age (young adult versus elderly population), by intervention components and by characteristics of outcome measures. Meta-regression analyses will be used to explore components of interventions, participant characteristics and outcome measure characteristics that can predict prevention effect estimates within and across different types of interventions. The network meta-regression will be performed by allowing for a common treatment-covariate interaction for each intervention in the network meta-analysis .To assess small study effects and publication bias, we will use funnel plots of pairwise meta-analyses if 10 or more studies are included. We will also use a comparison-adjusted funnel plot for relative treatment effects between all active and control interventions .In order to make different incremental cost-effectiveness ratios (ICERs) comparable, we will convert them from their currencies to pounds sterling (£) using purchasing power parities . Once coThe analysis and presentation of results will be performed using R  –36.The confidence in the relative treatment effect estimated in network meta-analysis for the primary outcome will be evaluated using the Confidence in Network Meta-Analysis framework , implemeDespite recent improvements in the burden of cardiovascular disease in the UK, deaths from cardiovascular disease are relatively high compared with other high-income countries. There is a major potential population health impact of improving our understanding of CVD prevention. Identifying the most effective intervention however remains a challenge for researchers and policymakers.This study will provide highly relevant findings using innovative methods to determine optimal strategies for primary prevention of CVD. The results will be important for policymakers when making decisions between the multiple possible alternative strategies to prevent CVD. Compared to results from the existing multiple separate pairwise meta-analyses, this overarching summary of all relevant work will enhance decision-making. The findings will be crucial to inform evidence-based priorities and guidelines for policies and planning prevention strategies. The public will have answers to which intervention or combination of interventions has the greatest probability of being most effective and most cost-effective in preventing CVD. Findings from our study will inform research funders and improve the targeting of subsequent research, and evaluation of additional population interventions.Given the paucity of randomised evidence on population-wide interventions, this project will assess data from observational studies which are more prone to potential bias. We will mitigate this by using Bayesian approach to coherently synthesise evidence from both randomised and non-randomised evidence, which will take into account the potential bias in the observational studies using an extra variance component.Findings from this study will be internationally relevant. Our findings will be widely shared with academics ; policymakers , stakeholders and key organisations such as the British Heart Foundation (executive summaries) and patient groups (lay summaries).Additional file 1. List of existing of existing systematic reviewsAdditional file 2. PRISMA-P ChecklistAdditional file 3. Cochrane CENTRAL search strategy