Chronic kidney disease (CKD) leads to significant morbidity and mortality worldwide. Glomerulonephritis (GN) is the second leading cause of CKD resulting in end stage renal failure. The most severe and rapidly progressive type of GN is characterized by glomerular crescent formation. The current therapies for crescentic GN, which consist of broad immunosuppressive drugs, are partially effective, non-specific, toxic and cause many serious side effects including infections, cancer, and cardiovascular problems. Therefore, new and safer therapies are needed. Human amniotic epithelial cells (hAECs) are a type of stem cell which are isolated from the placenta after birth. They represent an attractive and novel therapeutic option for the treatment of various inflammatory conditions owing to their unique and selective immunosuppressive ability, as well as their excellent safety profile and clinical applicability. In this review, we will discuss the immunopathogenesis of crescentic GN, issues with currently available treatments and how hAECs offer potential to become a new and harmless treatment option for this condition. The immune system and the kidneys are closely linked, and chronic kidney disease (CKD) often results from various auto(immune) disorders. CKD is due to slowly progressive, chronic deterioration of kidney function. Crescentic glomerulonephritis (GN) is a chronic immune-mediated disease which causes severe glomerular inflammation and injury, and often leads to irreversible kidney failure. It is a common cause of morbidity and mortality worldwide. GN is a major contributor to the escalating health burden associated with CKD.Almost all current therapeutic concepts in autoimmune diseases are based on the systemic suppression of immune functions and are not curative. Currently used immunosuppressive therapies for crescentic GN are only partially effective, toxic and provide broad, non-specific immunosuppression, thus producing significant adverse effects. These including mainly severe infections, but also cancer and cardiovascular events. It is these treatment-caused side effects which cause the majority of patient deaths. Therefore, new and safer therapies are much needed.Human amniotic epithelial cells (hAECs) are a type of stem cell which are isolated from the human placenta after birth. They are an attractive and novel therapeutic option for the treatment of crescentic GN due to their (i) ethical, non-invasive, and speedy isolation from the amniotic membrane of the placenta which results in an abundance of readily available cells, (ii) potent immunosuppressive capacity, (iii) low immunogenicity (ability to activate the immune system) and (iv) their ability to fight microbes and cancer and protect against cardiovascular disease. hAECs have attenuated various inflammatory diseases in mouse models, without being rejected, producing tumors or causing any major side effects. They are currently being tested in several clinical trials to treat different conditions including lung and liver disease and stroke. The purpose of this review is to discuss and link some of the major immune and inflammatory mechanisms to the progression of crescentic GN, discuss the common therapies and their limitations, and outline the therapeutic potential of hAECs in safely reducing glomerular injury.The term glomerulonephritis (GN) refers to immune-mediated inflammation of the renal glomeruli. GN is diagnosed based on clinical presentation, etiology, histopathology or pathogenesis. Most patients present with hematuria, proteinuria, and impaired glomerular filtration rate . This paCrescentic GN is a severe form of glomerulonephritis characterized by the destruction of the renal glomeruli that often lead to end-stage renal disease over a relatively short period of time  . It is cOn the basis of immunopathological findings, crescentic GN can be classified into three major categories: anti-glomerular basement membrane (GBM) antibody disease (Goodpasture’s syndrome), immune complex GN , and pauci-immune GN which is often associated with anti-neutrophil cytoplasmic autoantibodies (ANCA) and is thus also known as ANCA-associated vasculitis (AAV) . Anti-GBExperimental evidence for the pathogenesis of crescentic GN comes mainly from pre-clinical models, including lupus nephritis, experimental autoimmune GN , MPO-ANCA vasculitis and nephrotoxic nephritis (NTN).A large body of evidence about the immunopathogenesis of crescentic GN has come from one of the most widely used and best-characterized models, called nephrotoxic nephritis (NTN), also known as autologous anti-GBM globulin GN. In this model, rodents such as rats, mice and rabbits are passively injected with foreign polyclonal antibodies (globulins) targeting the mouse GBM . It compThe second phase is called the autologous phase and tends to begin about 6 to 7 days later. It is caused by an adaptive immune response to the foreign anti-GBM globulin . This phIn mice, an accelerated model of NTN can be induced by pre-immunizing mice with normal sheep globulin (NSG) . In acceOther models of crescentic GN exist and they have been also widely used to reveal pathogenetic mechanisms of disease. MPO-AAV can be induced in mice by immunizing animals with MPO which results in the development of active anti-MPO autoimmunity, followed by neutrophil lodgment in glomeruli and deposition of the autoantigen for subsequent recognition by MPO-specific T cells . AlternaMultiple immune mechanisms contribute to the pathogenesis of crescentic GN. A summary of the main immune pathways which positively or negatively regulate the development of crescentic GN is shown in Innate and adaptive immune system activation are a common underlying mechanism for several forms of crescentic GN . StudiesNumerous studies in various models of crescentic GN have shown that CD4 + T cells play a critical role in this disease . CD4 T hMoreover, it has become clear that the Th17 pathway is important in the development of crescentic GN. In experimental MPO-AAV, mice lacking IL-17A are protected from early glomerular injury . SimilarTregs are a specialized inhibitory subset of CD4 + T cells characterized by expression of CD25 and Foxp3. They play a key role as negative regulators of pathogenic immunity in crescentic GN. Studies using Foxp3-GFP reporter mice explored the functional role of T-regulatory cell in inhibiting anti-GBM nephritis . Foxp3 pIn addition to T cells, B cells and autoantibodies have also been demonstrated to play a major role in the development of crescentic GN. B cells and/or antibodies against various autoantigens including MPO in AAV, α3(IV)NC1 in Goodpasture’s and nuclear antigens in lupus, are pathogenic in experimental crescentic GN . In contIn human studies, strong evidence from patients’ kidney biopises, and blood samples has accumulated to support the role of immune cells and antibody in cresentic GN, with findings similar to those in animal models. While neutrophils are prominent in cresentic GN , a consiIn general, crescentic GN therapy is often classified into two phases. First is the activation of remission phase during the acute period, followed by the subsequent phase of maintenance therapy to control the underlying long-term immunopathology . The indCorticosteroids, synthetic drugs which closely resemble cortisol hormone, have been used for decades to modulate inflammation therapeutically by decreasing the movement of neutrophils to the inflammatory sites and inducing a transient lymphocytopenia . This meCombination therapy with pulse cyclophosphamide plus pulse methyl prednisolone therapy significantly reduce proteinuria, serum creatinine, the level cellular crescents in crescentic GN , and preHowever, induction therapy (corticosteroids with cyclophosphamide) is only partially effective, non-specific and produces many toxicities and serious side effects. Therefore, there is a major unmet need for safer, effective therapies. For example, in AAV, this treatment induces remission in 70–90% of patients, but the incidence of dialysis or death at 5 years is still high (∼30%), with the majority of early deaths caused by drug-related side effects, mainly infections caused by broad immunosuppression . In lupuAdalimumab, an anti-TNFα monoclonal antibody, has been used as an adjunct therapy to standard immunosuppression. It can be an effective therapy for the induction of remission in AAV and may permit reduced prednisolone dosing, thus decreasing therapy-related toxicity . HoweverMycophenolate mofetil (MMF), is a salt form of the immunosuppressive drug mycophenolic acid. It inhibits the releasing of proinflammatory cytokines, nitric oxide, and LDH in macrophages and suppresses proliferation and infiltration of both T and B lymphocytes . Recent Stem cell-based therapy is an attractive approach to ameliorate a broad range of human diseases and injuries, and it has been proven to be safe and effective in a wide range of immune-mediated diseases . Severalin vitro mal-transformation. It is also not optimal for the treatment of diseases such as crescentic GN in which early treatment is required to stop the progression of rapidly progressing deterioration of kidney function.Mesenchymal stem cells (MSCs) are multipotent adult stem cells that exist in various locations including umbilical cord, bone marrow and adipose tissue . MSCs haEmbryonic and induced pluripotent stem cells (ESCs and iPSCs) are pluripotent stem cells. ESCs are isolated from the inner cell mass of blastocysts, while iPSCs are produced from adult somatic cells that are genetically reprogrammed to an ESC-like state by transcription factors . It has One particular stem cell type, hAECs, have attracted much attention in the recent years as an ideal therapuetic option for the treatment of autoimmune and inflammatory disorders due to their immunosuppresive ability and their superior safety and clinical applicability over other stem cell types. hAECs are a heterogeneous epithelial population that originates from the lining of the inner membrane of the placenta which provides an abundant cellular source for stem cell-based therapy . In compHuman Leukocyte antigens (HLA), encoded by the major histocompatibility complex (MHC) gene complex in humans, are the major molecules that initiate graft rejection . HoweverTumorigenicity is a common obstacle for cell-based therapies, since some cells may cause formation of tumors due to being immortal. Unlike ESCs and iPSCs , hAECs din vitro studies showed similar inhibitory effects of hAECs on human cells. For instance, hAECs suppressed human CD4 + T cell proliferation, induced a Th2 cytokine profile, suppressed production of Th1 and Th17 cytokines and promoted differentiation of naïve CD4 + T cells into Tregs , which has several immunosuppressive properties such as inhibition of T cell proliferation. hAECs also secrete TGFb, a T cell growth inhibitor and a powerful immunosuppressive molecule  and SLE maintained organ function, minimized inflammation and modified the immune balance . In EAT,Human amniotic epithelial cell also potently attenuated disease severity in experimental autoimmune encephalomyelitis (EAE), and a mouse model of multiple sclerosis (MS) . T cell In an autoimmune uveitis (EAU) rat model, hAECs treatment ameliorated the pathological progression of disease and maintained the retinal structural organization . InfiltrThe immunomodulatory effect of hAECs has also been investigated in mice with autoimmune ovarian disease (AOD) . The outTransplantation of both hAECs and their soluble factors have shown beneficial effects in animal models of hepatic fibrosis. hAECs given to mice with induced liver fibrosis reduced hepatocyte apoptosis and decreased hepatic inflammation and fibrosis . This stIn a rat model of ischemic stroke, hAECs were administered by intracerebral injection, after which they reduced the infarct volume and cerebral apoptosis . A furthMany studies have explored the immunomodulatory effect of hAECs on lung fibrosis. A study using the bleomycin-induced model of pulmonary fibrosis showed that hAECs can modulate the host inflammatory response, decrease fibrosis and preserve lung function . hAECs rA recent study in a mouse model of renal ischemia-reperfusion injury, which leads to acute kidney injury, has shown that systematically administered hAECs effectively regulated the kidney immune response . hAEC inThe secreted factors  exist in the medium where the stem cells are cultured. Numerous studies on stem cell-derived secreted factors showed that these mediators alone, without the stem cell itself, affect the maturation, migration, polarization and function of immune cells, and thus influence the strength and duration of immune responses. The hAEC secretome contains metabolites, lipids, free nucleic acids, cytokines, growth factors and extracellular matrix proteins. These are all known to play crucial roles in cell-cell communication, acting proximally as well as systemically.2, after which conditioned media is harvested and secretome obtained by serial centrifugation  and patient deaths. hAECs, as well as hAEC-CM and hAEC-derived exosomes, exert a protective effect in models of various immune-driven conditions, with minimal side effects. They mediate their effects through multiple immunomodulatory and anti-inflammatory mechanisms, and their safety has been proven in clinical trials. Thus, hAEC-based therapy offers promise to be a safe, feasible and effective treatment for crescentic GN. This is due to their (i) ethical and speedy isolation from the placenta which results in an abundance of readily available cells, (ii) unique and selective immunosuppressive capacity, (iii) low immunogenicity, and (iv) ability to fight microbes and cancer and protect against cardiovascular conditions. If proven to be effective in pre-clinical models of crescentic GN, hAECs have the potential to change clinical practice in this disease and provide immense advantages to patients by alleviating their risk of death and complications from unwanted symptoms caused by the existing treatments.AA, JO, and DO wrote the article and agreed with the final version of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Drugs can be classified as hydrophilic or lipophilic depending on their ability to dissolve in water or in lipid-containing media. The predominantly lipophilic statins  can easily enter cells, whereas hydrophilic statins (rosuvastatin and pravastatin) present greater hepatoselectivity. Although the beneficial role of statins in primary and secondary cardiovascular prevention has been unequivocally confirmed, the possible superiority of one statin or other regarding their solubility profile is still not well-established. In this respect, although some previously published observational studies and clinical trials observed a superiority of lipophilic statins in cardiovascular outcomes, these results could also be explained by a greater low-density lipoprotein cholesterol reduction with this statin type. On the other hand, previous studies reported conflicting results as to the possible superiority of one statin type over the other regarding heart failure outcomes. Furthermore, adverse events with statin therapy may also be related to their solubility profile. Thus, the aim of the present review was to collect clinical evidence on possible differences in cardiovascular outcomes among statins when their solubility profile is considered, and how this may also be related to the occurrence of statin-related adverse effects. The classification of drugs as hydrophilic or lipophilic depends on their ability to dissolve in water or in lipid-containing media. In this respect, absorption is faster in lipophilic drugs, whereas the ease for renal excretion is greater in hydrophilic medications.As most drugs are weak acids or bases, in an aqueous solution they can be present in two phases: ionised or polar and non-ionised or non-polar. The ionised polar fraction is water-soluble whereas the non-ionised is fat-soluble and is the only one that diffuses easily through cell membranes. The degree of molecule ionisation depends on three main factors: their acidic/basic nature, the dissociation constant of the molecule measured by pK and the pH of the medium where it is found. However, the latter is the definite determinant of the drug's availability to cross cell membranes and act on diverse tissues. Moreover, the degree of digestive absorption and renal excretion of each drug can be modified by changing the pH of the medium where it is found .It must also be considered that hydrophilic substances can be excreted without undergoing any transformation. On the other hand, although the kidney can poorly filter ionised molecules (lipophilic), these are mostly reabsorbed back into the tubule. For this reason, most lipophilic substances are metabolised to become more polar metabolites, which then become water-soluble.Thus, the hydro- or liposolubility of each drug is an aspect that should be carefully considered in clinical practise when deciding on the type of treatment, together with dose adjustment in patients with renal failure. In that clinical scenario, lipophilic drugs should be avoided, although other clinical characteristics must also be considered.Since the introduction of lovastatin in 1987 as the first 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor approved for human therapy, statins have become the most widely used lipid-lowering drugs with proven effect in cardiovascular disease prevention in different clinical settings –5. StatiStructurally, statins have three main parts as detailed in Sufficient evidence of the possible beneficial or harmful effects related to statin solubility is lacking. It has been speculated that the ability of lipophilic statins to reach extrahepatic tissues could account for the more favourable cardiovascular outcomes in subjects receiving this type of lipid-lowering drug, although with a higher risk of adverse effects such as statin-associated muscle symptoms (SAMS).The present comprehensive review aimed to describe the clinical evidence available to date regarding the possible differences in cardiovascular outcomes among statins when their solubility profile is considered, and how this may also be related to statin-related adverse effects. We hope this review will aid better understanding of statins for physicians and ultimately be useful for precise statin selection since, though they share the same mechanism of action, different chemical and pharmacological characteristics may affect their therapeutic efficacy.in vitro and experimental studies, it is not possible from available clinical evidence to isolate the potential benefits of pleiotropic effects of statins from those conferred by LDL cholesterol reduction.Statins inhibit cholesterol synthesis, thereby enhancing LDL clearance from the circulation. Since mevalonic acid is the precursor of numerous metabolites, HMG-CoA reductase inhibition potentially results in pleiotropic effects that may affect several tissue functions and modulate specific signal transduction pathways . These eOne of the factors to be considered when evaluating outcomes of lipid-lowering treatment with statins is their solubility. Therefore, the role of both hydrophilic and lipophilic statins regarding beneficial pleiotropic effects and thus a possible improvement in cardiovascular primary or secondary prevention needs to be analysed further.According to the 2016 European Society of Cardiology definition , heart fThe role of statin therapy in HF remains controversial, with conflicting findings from observational studies and clinical trials , 13. NexSome trials (including both hydrophilic and lipohilic statins) on cardiovascular prevention reported interesting findings in the HF field. In this respect, those which compared statin vs. placebo , 15 or mA large-scale meta-analysis of randomised primary and secondary cardiovascular prevention clinical trials with statins showed a modest reduction (10%) in first non-fatal HF hospitalisation with statin therapy, with no effect on HF death. However, no differences were found in risk reduction between patients who presented an incident myocardial infarction or not. Only 10–15% of non-fatal HF hospitalisations were preceded by a documented within-trial non-fatal myocardial infarction . AlthougMore recently, Imran et al. , evaluatIn summary, more evidence is needed to support the use of high-intensity hydrophilic statins in the context of incident HF prevention.Data from the major lipid-lowering trials on the effects of statin therapy on prevalent HF are scant since the majority excluded patients with this syndrome , 23.Beside small studies with atorvastatin (lipophilic) –26 or otp = 0.01], cardiovascular mortality  and hospitalisation for worsening HF ; however, both statin types were comparable for cardiovascular hospitalisation . Similarly, other studies also showed a greater risk reduction in HF events, hospitalisation and mortality  with lipophilic vs. hydrophilic statins  Collaborators of statin treatment in people at low cardiovascular risk demonstrated a 9% reduction  in all-cause mortality and 25%  in major vascular events per 1.0 mmol/L reduction in LDL cholesterol, even among low-risk patients . FurtherWhether the solubility profile of each statin type could account for this favourable cardiovascular benefit remains open to debate. In this respect, the previously mentioned CTT meta-analysis  includedThe possible differences between hydrophilic and lipophilic statins have mainly been evaluated regarding secondary cardiovascular prevention in patients with acute coronary syndrome and stable CHD  52–59).–59.52–59Back in 2004, the PROVE IT-TIMI 22 trial  comparedp = 0.001; 6 months: 19.9 vs. 14.2%; p = 0.022), whereas no significant difference in MACE was observed at 1 year of follow-up . Both statin arms showed similar efficacy in the lipid profile and the use of a hydrophilic statin did not predict 1-year MACE, all-cause death, acute myocardial infarction or re-percutaneous coronary intervention.Kim et al.  found a p = 0.0069), acute coronary syndromes tended to occur less frequently  and the incidence of new a Q-wave appearance on electrocardiogram was significantly lower  with hydrophilic pravastatin than with lipophilic statins. The CENTAURUS trial (p = 0.02), although these results were not further confirmed with a longer follow-up. Finally, Izawa et al. (p < 0.001 for each).To shed light on this matter, further trials such as that reported by Sakamoto et al.  in 2007 a et al.  obtainedp = 0.682), myocardial ischaemia , cardiovascular death  and all-cause mortality . However, the cardiovascular hospitalisation rate was lower  and alanine aminotransferase (ALT) elevation higher  in lipophilic- than in hydrophilic-treated patients.On the other hand, the findings of Bytyçi et al.  between p = 0.02). However, as in the CTT meta-analysis (p < 0.001), and hence the solubility profile of each statin and the possible superiority of lipophilic statins could play a secondary role in the observed differences.With regard to coronary atherosclerosis progression/regression studies, the REVERSAL trial  assessedanalysis , this cop < 0.001 for each treatment group) with no significant difference between treatment groups. However, atorvastatin-treated patients had greater LDL cholesterol reductions than pravastatin-treated patients, a trend towards fewer MACE  and a significantly greater reduction in all-cause death . Hence, as previously mentioned, the probable superiority of lipophilic atorvastatin could once again be explained by its greater potency in lowering LDL cholesterol concentrations.Moving forward, it must be acknowledged that the previously mentioned studies focused mainly on acute coronary syndrome. Here, the possible beneficial effects of one statin type or the other (hydrophilic vs. lipophilic) may probably depend more on their pleiotropic effects since the impact of lipid-lowering therapy in LDL cholesterol reduction has not yet been attained. However, when the focus is on chronic ischaemic heart disease, the beneficial effects of LDL cholesterol reduction may be present to the same degree as the pleiotropic changes. In this respect, Deedwania et al.  evaluateFinally, the possible pleiotropic effects that may account for all these observed results include decreased adenosine triphosphate (ATP) production with lipophilic statins and enhanced myocardial stunning after ischaemia and reperfusion , with diNevertheless, while some studies, particularly randomised controlled trials, detected superiority of hydrophilic statins regarding to secondary CHD prevention, others reported greater LDL cholesterol reductions with lipophilic statins, which could also account for the more favourable cardiovascular outcomes. As for HF outcomes, we believe future randomised trials with longer follow-up are mandatory to confirm the possible superiority of one statin type over the other taking into account their solubility profile, and regardless of their intensity in lowering LDL cholesterol levels.The possible differences between statin types and the risk of atrial fibrillation related stroke has also been evaluated. In this sense, a meta-analysis  includinAlthough the different statin types have possible beneficial effects depending on their solubility profile, safety cannot be ignored. It has been argued that the benefits of lipophilic statins may transcend into diverse adverse reactions owing to their easy penetration into extrahepatic tissues. However, solid evidence is still lacking in this field , 28, 68.Firstly, the prevalence of SAMS differs between statin classes. Lipophilic statins such as simvastatin, atorvastatin, fluvastatin, pitavastatin and lovastatin, owing to their well-known ability to non-selectively diffuse into extrahepatic tissues, such as skeletal muscle, carry a higher risk of SAMS. In contrast, hydrophilic statins have less muscle penetration and therefore lower risk . HoweverSecondly, the presence of new-onset diabetes mellitus with statin therapy should also be mentioned. This seems to be more frequent in patients with pre-existing risk factors, including metabolic syndrome traits . It has per se but rather result from changes in cholesterol concentrations.As for neurological disorders, it has been hypothesised that lipophilic statins could induce a higher risk given their increased ability to cross the blood-brain barrier ; howeverFinally, controversy persists regarding the effects of statins on renal function. Except for hydrophilic pravastatin and rosuvastatin, the remaining statins are mainly metabolised by the liver and minimally cleared by the kidney. Mild transient proteinuria has previously been observed in some patients when receiving high-dose statin treatment; however, this has not been firmly associated with impaired renal function .The classification of drugs as hydrophilic or lipophilic depends on their ability to dissolve in lipid media or in water. The predominantly lipophilic statins can easily enter cells and interact with cell membranes, whereas hydrophilic statins present greater hepatoselectivity.Conflicting results have been observed on the superiority of hydrophilic or lipophilic statins regarding cardiovascular outcomes, including HF and CHD, both from primary and secondary prevention. In this respect, the possible superiority of lipophilic statins seen in some studies could be explained by a greater LDL cholesterol reduction with this statin type, with the solubility profile playing a secondary role in the favourable cardiovascular outcomes observed.Finally, the non-selective diffusion of lipophilic statins into extrahepatic tissues could account for an increase in SAMS, albeit without differences between hydrophilic and lipophilic statins with respect to other adverse effects. Thus, we believe future studies are essential in this field for the solubility profile of statins to be taken into account when deciding on the optimal lipid-lowering therapy for each patient in daily clinical practise.The first version of the manuscript was written by EC, and revised and approved by DB and JP-B. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recombinant adeno-associated viral vectors (rAAV) represent a gene therapy tool of ever-increasing importance. Their utilization as a delivery vehicle for gene replacement, silencing and editing, among other purposes, demonstrate considerable versatility. Emerging vector utilization in various experimental, preclinical and clinical applications establishes the necessity of producing and characterizing a wide variety of rAAV preparations. Critically important characteristics concerning quality control are rAAV titer quantification and the detection of impurities. Differences in rAAV constructs necessitate the development of highly standardized quantification assays to make direct comparisons of different preparations in terms of assembly or purification efficiency, as well as experimental or therapeutic dosages. The development of universal methods for impurities quantification is rather complicated, since variable production platforms are utilized for rAAV assembly. However, general agreements also should be achieved to address this issue. The majority of methods for rAAV quantification and quality control are based on PCR techniques. Despite the progress made, increasing evidence concerning high variability in titration assays indicates poor standardization of the methods undertaken to date. This review summarizes successes in the field of rAAV quality control and emphasizes ongoing challenges in PCR applications for rAAV characterization. General considerations regarding possible solutions are also provided. Recombinant adeno-associated virus (rAAV) is an increasingly important gene therapy vector. These vectors became popular due to their natural characteristics. First of all, wild-type AAVs are not associated with any human disease. At the same time, naturally occurring AAV serotypes are able to transduce different tissue and cell types in vitro and in vivo, and their categorization has expanded with creation of engineered capsids. AAVs’ replication deficiency without a helper virus, as well as their low immunogenicity, prolonged transgene persistence and low integration efficiency into known and safe location makes them even more attractive delivery vehicles ,2.To date, three AAV-based therapies have been approved for medical application: Glybera , Luxturna (voretigene neparvovec) and Zolgensma (onasemnogene abeparvovec). These products utilize rAAV serotypes 1, 2 and 9 for the delivery of a functional gene copy. Gene delivery by viral vector can either compensate for a malfunctioning gene (gene replacement therapy) or provide a new function to help fight a disease (gene addition therapy). Collectively, these gene therapy approaches are being tested in more than 200 clinical trials . AnotherNumerous clinical trials of rAAV-based therapeutics, vaccine development and laboratory applications of vectors, require the production of diverse rAAV preparations in large quantities. Differences can rely on either capsid proteins or internal effector and regulatory sequences. Scientists generate engineered capsids aimed to deliver genetic constructs to distinct tissue and cells, increase vector circulation time and reduce the titer necessary for infection. Gene therapy strategies encompass more diseases and require the delivery of new genes and regulatory nucleic acids. Continuous modifications of existing expression cassettes and the introduction of new regulatory elements aim to control expression inside the target tissue, along with minimizing undesirable adventitious effects. Variability in resulting capsids and genetic constructs raises the question of standardized methods regarding rAAV analytics, primarily quantification. Titer measurement methods encountering viral particle (vp) and viral genome  number should be distinguished from methods estimating infectious titer. The first group combines ELISA, dot blotting and others to detect capsids, while the second uses PCR-based methods and nucleic acid staining without amplification. Cellular methods are utilized to estimate the number of infectious units. Wide characterization of rAAV preparations provide information about quantity, identity, stability, functionality and presence of substances that may potentially interfere with successful vector administration . The majThe growing popularity of rAAV vectors, not only as gene therapy vehicles but as experimental tools, compels researchers to examine multiple methods for their quantitative and qualitative analyses. In this review, we focus on PCR-based methods for rAAV quality control (QC). The final product characteristics mainly depend on AAV nature and rAAV production method; thus, the next two sections are devoted to these issues. An adeno-associated virus is a small (~25 nm) parvovirus that contains a single-stranded DNA (ssDNA) with a genome length of 4.7 kilobases, enclosed in a protein capsid . The wilAAV tropism to the target tissue is explained by a combination of tissue-specific receptors present on the cell plasma membrane. For example, AAV2, AAV3B and AAV6 attach to heparin sulfate proteoglycan. Glycan-conjugated sialic acid is known to interact with AAV1, AAV4, AAV5 and AAV6. AAV9 has a preference for glycans ending in a terminal galactose. Necessary co-receptors have also been reported: the hepatocyte growth factor receptor and human fibroblast growth factor receptor-1 for AAV2; platelet-derived growth factor receptor for AAV5; integrin α5β1, LamR, AAVR and GPR108 for a variety of serotypes . FollowiThus, AAV serotypes have different capsid protein sequences, impeding development of a universal method for titration based on recognition by specific antibodies. Encoding sequences vary from one sample to another, but exhibit common features, such as similar length and obligatory presence of ITRs. The simplicity of PCR methods’ customization for transgenes is one reason for the popularity of these methods in the field. Moreover, targeting the ITR sequence during qPCR allows for the detection of a wide variety of rAAV preparations using the same protocol.Recombinant AAV production methods differ between research groups. The basic principle of rAAV production implies three genetic constructs delivered to producer cells. The first construct contains the gene of interest flanked with ITRs. The second bears rep/cap sequences and defines serotypes of assembled rAAV. The third introduces helper genes, necessary for AAV genome replication. Depending on the particular production platform, helper factors may be derived from plasmids, helper viruses or stable transformed cell lines. Role of helper virus can be efficiently carried out by adenovirus, introducing E1A, E1B, E2A and E4ORF6 genes, and VA RNA or herpes simplex virus type 1 (HSV-1), providing UL5, UL8, UL52 and UL29 factors, essential for AAV production. Several other viruses, such as members of herpesvirus family, papillomaviruses, as well as baculovirus and human bocavirus 1 are also known to have helper function, wherein their efficacy may be reduced . DespiteThe platform used for rAAV sample production should be taken into account during the development of quantitation and quantification methods, as rAAV characteristics and contaminating agents can differ significantly. For example, the Sf9 insect cell production platform is known to generate a higher percentage of empty and dysfunctional rAAV particles in comparison with other methods. Moreover, Sf9 cells propagate viral particles with different capsid protein modifications and genome methylations than mammalian producer cell lines . On the To conclude, AAV production systems introduce another challenge for quantification and QC of viral preparations, necessitating many tests to sufficiently characterize different products in terms of safety and efficiency.Quantitative assays provide information about vector titer as one of the most important QC criteria. Titer estimation in crude viral lysates allows us to assess the assembly efficiency of a newly designed vector construction and follow the changes in this parameter after vector genome or capsid modifications. During purification, total rAAV quantity shows efficiency at a particular stage. Accurate quantification in final rAAV preparation is important for applying the known number of vector particles in further tests. The last parameter is of utmost importance for rAAV therapies or preparations undergoing preclinical and clinical trials, since the applied vector dose will determine therapeutic efficacy and product safety . To dateThe encapsidated AAV genome is a key component of the vector that mediates the transfer of the transgene and, therefore, the functional effect. Titration of vector genomes by quantitative real-time PCR (qPCR) became a widely accepted method for rAAV quantification and clinical dose determination ,31,32. AQuantitative real-time PCR is a type of PCR that enables the detection and measurement of products generated in each cycle of the amplification process. Detection is based on a fluorescence signal that is emitted by DNA-intercalating dyes or fluorescently labeled target-specific probes. The number of cycles when fluorescence intensity crosses the threshold is proportional to the amount of target template added to the reaction . To calcMeasuring AAV titer by means of qPCR was first reported in the late 1990s . Since tFirst protocols of qPCR-based AAV titering assays were designed to target specific sequences of recombinant AAV genomes. CMV promoter-specific primers were reported by Rohr and colleagues . The traA versatile quantitative PCR protocol, targeting conservative sequences in the rAAV genome, could solve the problem of standardization. The only fairly conserved sequences of viral origin in rAAVs are ITRs. Together with this fact, pseudotyping, a major strategy of recombinant AAV genome designing, makes ITR an ideal candidate as a universal qPCR target. AAV2 was the first serotype to be converted to a vector and has since become the most widely studied serotype, especially in preclinical and clinical trials, before other serotypes became available ,41,42. CThe versatility of ITR-based qPCR appears to be its main advantage over transgene- or regulatory element-specific titration qPCR assays. However, accumulating data regarding the application of this system to a number of AAV vector preparations revealed its susceptibility to inadequate titer estimation. Depending on the research group, titers defined by ITR-targeting qPCR were underestimated ,53 or ovConsidering the mentioned inadequacy in titration, ITR-based PCR quantification of AAV vectors requires additional controls for undistorted titer estimation. Nevertheless, the versatility of the method makes it attractive for researchers.Sample pretreatment is an important part of titration protocols. The first indispensable step is DNase I treatment. This prevents titer overestimation by amplifying non-encapsidated DNA, originating from transfection plasmids or disrupted capsids. Such pre-treatment is even more important when intercalating dye, such as Sybr Green, is used for amplified DNA measurement . To moniVector pre-treatment improvements may be continued with the addition of a proteinase K digestion step. Such a modified protocol was described, for instance, by Lock and colleagues . AlthougAnother group of studies aimed to prevent material loss due to adherence to various materials during titration, particularly at low concentrations. Usually, this includes the addition of surfactant Pluronic F-68 to the buffer used for the dilution of analyzed samples and calibration standards. Utilization of Pluronic F-68 conferred a significant increase in titers, and markedly increased vector quantification accuracy ,58. NotiAs well as an increase in variability, additional pre-treatment steps may lead to an increased risk of cross-contamination of samples . SubjectDespite proposed protocol modifications aimed at minimizing the titer variability discussed above, utilizing a standardized titration protocol is not be the only requirement for comparable results. This is evidenced in a large interlaboratory study, performed by AAVRSM  Working Group during wide characterization of AAV reference materials of serotypes 2 and 8 ,59. FinaUtilization of different DNA types with known concentrations as a calibration standard can significantly impact rAAV genome quantitation. Standards for rAAV titration can be presented by plasmid DNA, linear DNA fragments, purified viral genomes and viruses themselves. The main attributes of a good standard include the resembling secondary structure and stability to obtain precise and reproducible measurements. Different types of standard sample amplification errors may result in both overestimation and underestimation of rAAV samples. Possible variants are demonstrated on A distinct type of linearized vector plasmid, the so-called “free-ITR” calibration standard, deserves special attention ,49. GeneEstablished calibration plasmid standards, as discussed above, share poor structural similarity when analyzing self-complementary vector genomes, and therefore provide inconsistent results . Thus, sFinally, denatured rAAV  and enti14 GC, measured by qPCR with supercoiled plasmid as calibration sample, appeared to be equivalent to 1.1 × 1014 GC measured by ddPCR [The importance of choosing calibration standards for accurate dosage measurements of rAAV-based gene therapy is shown in ongoing clinical trials. Substitution of rAAV titration methods during clinical studies of Zolgensma (AveXis) for spinal muscular atrophy treatment led to dose correction. Initial dose 2 × 10by ddPCR . During by ddPCR .Unrelated to the chosen type of calibration standard, internal reference rAAV material should be added to each plate when analyzing samples to minimize the interassay variability and to make interlaboratory comparisons viable. This primarily refers to plasmid DNA-based calibration standards due to their lower stability. An additional internal reference sharing maximal structural identity with analyzing AAV vectors may be recommended for the mentioned calibration systems utilizing degraded rAAV or purified rAAV genomes, where DNA is, also, unprotected from degradation. Currently, such references are available for AAV serotypes 2 and 8 from ATTC and other commercial laboratories. However, each laboratory can at least prepare and use their own reference material. For this reason, a highly purified rAAV batch with a known titer should be formulated, aliquoted and properly stored under constant conditions to ensure rAAV stability ,65.Droplet digital PCR (ddPCR) has recently emerged as a powerful technique for the absolute quantification of AAV. This method utilizes the same chemistry for the detection of amplified target sequences as qPCR, such as DNA-intercalating dyes or fluorescently labeled target-specific probes; thus, it allows for the direct transfer of amplification protocol from qPCR to ddPCR. This approach’s differences in relation to traditional analog PCR lie in sample pre-processing prior to amplification. Rather than amplifying DNA from bulk samples, in droplet digital PCR, individual DNA fragments are partitioned into unique droplets, in which the amplification reactions occur. By partitioning, digital PCR makes it possible to directly count the number of DNA molecules within an initial sample without requiring calibration standards. Another benefit of this method is that, unlike qPCR, the number of AAV vector genomes determined by ddPCR is less affected by the primers and probes used, and it is also less sensitive to inhibitors originating from components in the formulation . This arDigital PCR (dPCR) was first introduced in the 1990s ,69, but In recent years, ddPCR has become the standard technique for industrial use due to its advantages over traditional qPCR techniques, including reduced susceptibility to the presence of impurities and the capacity for absolute quantification without a standard curve . However11 gc/mL [In addition to PCR-based methods, other techniques can be applied for rAAV genome copy number estimation. For example, direct staining of viral samples in agarose gels with fluorescent dyes was proposed, with the aim of determining titers of rAAV without genome amplification . This me11 gc/mL . Despite11 gc/mL .Another group of methods unites DNA dot/slot blot and Southern blot . BrieflyUV spectrophotometry is another simple assay that is applied in rAAV vector genome quantifications . Viral vTaken together, non-PCR-based methods of rAAV titration either struggle to provide acceptably low levels of variation or show non-sufficient specificity and are prone to inaccuracy. However, such assays fit perfectly in the initial characterization of newly-generated vectors.Residual DNA impurities may pose safety concerns for vector preparations. One should pay attention to both free and encapsidated DNA, which is resistant to nucleases during purification and resembles target rAAV particles. Immunogenicity may arise due to induction of innate immunity by free DNA fragments themselves  or due to the expression of exogenous viral or plasmid DNA, whose products may be recognized by Toll-like receptors . EncapsiDepending on the production system used, viral oncogenes present within producer cells, such as adenovirus sequences E1 and SV40 large T antigen from HEK293(T) cells or human papillomavirus sequences E6 and E7 from HeLa cells, may remain as impurities in the final vector preparation ,78. ImpoDuring assembly of rAAV vectors, ITRs function as a key packaging signal. However, the non-vector sequences listed above can also be packaged; this could happen if rAAV vector recombination with non-vector DNA occurs. This process is driven via both homologous and non-homologous recombination. Thus, AAV vector preparations can contain a variety of chimeric sequences that consist of a vector ITR attached to non-vector sequences. A major concern is related to possible recombination between ITR-containing and rep-containing constructs, resulting in replication-competent AAV (rcAAV) particles. They can express rep and cap genes, leading to immunotoxicity, or replicate in the presence of helper viruses . SequencThe presence of DNA impurities can be quantified by PCR-based systems with primers and probes specific to contaminating sequences  73,87,88,89. ForThe amount of encapsidated DNA impurities in rAAV products is highly dependent on the vector design and manufacturing method. For instance, for scAAV manufacturing by transient transfection of plasmids in human cells, up to 26% of total capsids containing illegitimate DNA was reported ; whereasAnother issue concerns the inability to evaluate the presence of a truncated genome. Incomplete forms of vector genome may form during rAAV replication, especially in the case of genomes with strong DNA secondary structures or due to capsid destruction events ,110. ThePCR methods are widely used for both quantitative and qualitative analysis of rAAV preparations. However, one should critically assess rAAV samples only by their combination with other methods, especially when comparing viral preparations obtained from different production platforms, downstream processing, formulation and storage, taking into account possible impurities and degradation products . QC straOne should keep in mind that an equal genome copy number can be measured in samples with different full/empty ratios. As a result, when used in animal experiments, different total capsid contents can influence target cells’ transduction efficiency and immunotoxicity . This caAnother example that can affect a sample’s characteristics is measuring the gc number without assessing the percentage of aggregated viral particles. Aggregates can appear in highly concentrated viral samples with low ionic strength and as a result of violations in purification protocol or storage conditions . AggregaSome of the QC methods noted above can be set up using commercially available kits and reference samples. Others should be developed for new viral preparations in a sequence- and product-specific manner. During the development of QC methods, researchers should address critical parameters such as linear range, detection limit and susceptibility to specific and non-specific impurities. The majority of methods used for rAAV quantification and QC demonstrate high sensitivity and repeatability in pure viral samples; however, the purification step is not necessary for some applications  requirinRecent advances in the field of rAAV product approval for medicine applications suggest that current analytical methods are able to sufficiently characterize vector preparations to demonstrate the safety and efficacy of rAAVs in regulatory agencies .A rapidly growing number of clinical trials have highlighted the need to optimize the production of AAV vectors and subsequent processing as cost, among other reasons, led to product discontinuation shortly after the first approval of AAV-based gene therapy . CharactPCR methods are also widely used for the QC of rAAV preparations in terms of process-related impurities that combine host cell products, genetic constructs and contaminating viruses’ quantitation; product-related impurity assessments, which include mispackaged sequences of different origins, are also vital. The key point of PCR methods is the detection of truncated viral genomes, which can be recognized by the majority of PCR-modifications but remain non-functional. For this purpose, qPCR techniques can be accompanied by high throughput sequencing (HTS) applications. Currently, HTS-based methods for the assessment of DNA-related impurities demonstrate higher sensitivity than conventional QC tests based on real-time PCR , as wellThe development and adaptation of ddPCR has revolutionized rAAV quantification and characterization, demonstrating high reproducibility, low sensitivity to impurities and eschewing calibration samples. However, the availability of the necessary equipment and the cost of the procedures preclude many laboratories from deviating from traditional quantitative PCR methods, despite ddPCR and HTS methods representing a considerable development on their predecessors.


Tumor-derived exosomes (TEX) present in all body fluids of cancer patients, are considered promising candidates for a liquid tumor biopsy. TEX also mediate immunoregulatory activities: they maintain a crosstalk between the tumor and various non-malignant cells, including immunocytes. Effects that EVs exert on immune cells may be immunosuppressive or immunostimulatory. Here, we review the available data for TEX interactions with immunocytes, focusing on strategies that allow isolation from plasma and separation of TEX from sEV produced by non-malignant cells. Immune effects mediated by either of the subsets can now be distinguished and measured. The approach has allowed for the comparison of molecular and functional profiles of the two sEV fractions in plasma of cancer patients. While TEX carried an excess of immunosuppressive proteins and inhibited immune cell functions in vitro and in vivo, the sEV derived from non-malignant cells, including CD3(+)T cells, were variably enriched in immunostimulatory proteins and could promote functions of immunocytes. Thus, sEV in plasma of cancer patients are heterogenous, representing a complex molecular network which is not evident in healthy donors’ plasma. Importantly, TEX appear to be able to reprogram functions of non-malignant CD3(+)T cells inducing them to produce CD3(+)sEV enriched in immunosuppressive proteins. Ratios of stimulatory/inhibitory proteins carried by TEX and by CD3(+)sEV derived from reprogrammed non-malignant cells vary broadly in patients and appear to negatively correlate with disease progression. Simultaneous capture from plasma and functional/molecular profiling of TEX and the CD3(+)sEV fractions allows for defining their role as cancer biomarkers and as monitors of cancer patients’ immune competence, respectively.Extracellular vesicles (EVs) play a key role in health and disease, including cancer. Tumors produce a mix of EVs differing in size, cellular origin, biogenesis and molecular content. Small EVs (sEV) or  Small extracellular vesicles (sEV), otherwise known as exosomes, have recently emerged as potential biomarkers in various human diseases ,2. In caExosomes are a subset of extracellular vesicles (EVs) that differ from other EVs by their size, origin and molecular content. While the nomenclature for EVs has not been formally established, there is an agreement that small EVs (sEV) are distinct from larger EVs ,10. ExosMuch of what is currently known about TEX is based on analyses of EVs present in supernatants of cultured tumor cell lines ,21,22,232 (PGE2) and interfered with the generation of cytolytic T cells . Us. UsnonTE therapy . Another therapy . Thus, o921 . TEX therapy ,35,82. AThe remarkable recent progress in immunotherapy of solid cancers with ICIs has called attention to a long-neglected role of the host immune system in regulating cancer progression and response of patients to oncologic therapies. It is well documented that advanced metastatic disease is associated with profound immune dysfunction, which is characterized by high numbers of circulating immunosuppressive sEV. In this review, we have summarized the evidence showing that these circulating sEV, including TEX and non-TEX, carry and deliver messages relevant to patients’ immune competence and thus to tumor activity, stage, grade and migration capability. Immunosuppressive sEV circulating in cancer patients’ body fluids regulate anti-tumor responses of immune effector cells and thus exert impact on disease progression and on patients’ responses to oncologic therapies. The quality and quantity of molecular messages sEV carry can now be measured in TEX and non-TEX fractions, serving as potential cancer as well as immune competence biomarkers. Extensive reprogramming by TEX of non-malignant cells in the TME represents a vicious cycle that leads to ever greater immune suppression mediated by TEX and non-TEX produced by reprogrammed immune cells. Our data suggest that TEX and sEV produced by non-malignant cells have a potential to qualify as non-invasive biomarkers of cancer progression and response to therapy. These data are preliminary and require validation in future experiments and in clinical trials. An opportunity for capturing TEX and CD3(+) sEV from the same plasma specimen as described above, extends the list of potentially informative messages plasma sEV carry in their cargo. Currently, the challenges of fitting or decoding the messages in sEV cargo and correlating them with clinical information remain the major aspect of the ongoing biomarker validation process. We are confident that technical challenges of working with sEV will be successfully solved in the near future and that TEX and non-TEX serving as a liquid biopsy will contribute to cancer diagnosis, prognosis and monitoring responses to oncological therapies.


Many individuals in need of mental health services do not currently receive care. Scalable programs are needed to reduce the burden of mental illness among those without access to existing providers. Digital interventions present an avenue for increasing the reach of mental health services. These interventions often rely on paraprofessionals, or coaches, to support the treatment. Although existing programs hold immense promise, providers must ensure that treatments are delivered with high fidelity and adherence to the treatment model. In this paper, we first highlight the tension between the scalability and fidelity of mental health services. We then describe the design and implementation of a peer-to-peer coach training program to support a digital mental health intervention for undergraduate students within a university setting. We specifically note strategies for emphasizing fidelity within our scalable framework, including principles of learning theory and competency-based supervision. Finally, we discuss future applications of this work, including the potential adaptability of our model for use within other contexts. Mental illness is a pressing and growing global public health crisis with enormous societal costs . BetweenOne widely studied scalable approach, used most prominently in low-resource contexts, is for paraprofessionals to provide or support the delivery of scalable mental health services ,9. In thDigital mental health innovations via phone, computers, and other electronic devices offer another pathway for increasing access to care . Digitalscalability, to maximize intervention reach, with fidelity, to optimize quality and standards of treatment delivery. Scalability can be defined as “the capacity of an intervention to be applied in a way that reaches a large number of people” [Accordingly, mental health care models that combine paraprofessional workforces and digital mental health innovations have unique potential to expand the reach of and engagement with high-quality EBTs. One key consideration in efforts to design and implement paraprofessional-supported digital mental health interventions involves balancing  people” . Fidelit people”  to ensur people” .The purpose of this paper is to demonstrate 1 way of designing a coaching program that maintains a focus on the fidelity and delivery of high-quality EBTs, while preserving key strengths of paraprofessional models of care, including scalability. Our program was developed to support the delivery of a digital mental health intervention  on colleParaprofessional models have gained widespread attention and support as scalable models of mental health service delivery with great potential to address unmet needs for care ,24. EvidFidelity-monitoring practices have the capacity to increase therapist accountability in service of promoting treatment adherence and competence . Indeed,The financial and human resources needed to support fidelity monitoring in real-world contexts are often not available, limiting the external validity of many fidelity-monitoring strategies typically used in clinical trials . Even whDigital therapies hold significant promise for addressing problems with fidelity and bridging gaps in care access within wide-scale implementation efforts ,30. In pOne consideration in designing paraprofessional models is who should be trained to provide, or support the delivery of, mental health interventions. A prominent model focuses on training of peer-to-peer specialists, or peer coaches . Peer coYet, a major barrier to broader implementation of peer support is the mixed empirical support for these models -49. TherIn 2015, the University of California, Los Angeles (UCLA) launched a campus-wide research initiative, the Depression Grand Challenge (DGC), with the goal of cutting the burden of depression in half by 2050. The DGC comprises a number of studies that seek to uncover mechanisms underlying depression and to develop novel treatments and innovative approaches to treatment implementation. To begin tackling this problem at UCLA, the DGC launched the Screening and Treatment for Anxiety and Depression (STAND) program for UCLA students in fall 2017 . The STASTAND Digital Therapy is a modular program that combines interventions for depression, sleep, panic/agoraphobia, social anxiety, worry , and trauma (posttraumatic stress disorder), drawing upon existing evidence-based programs -66. TherFitting within this model, the initial development of our coach training program specifically targets UCLA undergraduate students as both coaches and recipients of the intervention, consistent with the peer support models described before. Enrollment as a coach trainee does not rely on any prerequisite coursework, history of service provision, or experience of personal mental health concerns or psychotherapy. Training and supervision of coaches are provided by graduate students in the clinical psychology doctoral program at UCLA for all stages of coach training. Graduate supervisors attend group supervision-of-supervision with a licensed clinical psychologist (author EGG).In our program, coach training occurs in weekly sessions, wherein trainees review digital CBT content, engage in didactic instruction of coaching materials, and complete role-play exercises focusing on basic interpersonal process skills. Coaches move through 4 primary phases of training: (1) beginner, (2) intermediate, (3) advanced, and (4) certified. Weekly training consists of a 2-hour training session as well as 2 hours of assignments completed between training sessions. Each level of training is completed over 1 academic quarter (10 weeks), at which point trainees are advanced to the subsequent level of training based on supervisor evaluations.The goals of the beginner phase of training are to (1) introduce coaches to digital CBT content and increase knowledge of the intervention and (2) provide early practice with interpersonal process skills to initiate the process of translating declarative knowledge during coaching delivery. In service of these aims, beginner-level trainees enroll as users of the digital CBT and advance through the digital CBT content themselves, completing homework exercises associated with the program and reading foundational material on cornerstone CBT topics between didactic training sessions. In addition, beginner-level trainees are introduced to 6 core interpersonal process skills that are routinely assessed to monitor coaching effectiveness throughout the coach training program: (1) authenticity, (2) nonverbal skills, (3) open-ended questioning, (4) reflecting emotions, (5) content summaries, and (6) collaborative inquiry -69. ThesBeginner-level trainees participate in (1) didactics regarding digital CBT content and interpersonal process skills, (2) discussions regarding other cornerstone topics , and (3) role-play exercises to begin practicing application of the 6 core interpersonal process skills. Beginner trainees also attend sessions with advanced trainees, in which they serve as mock or practice participants for advanced trainees who are coaching full mock sessions (described in detail in the Advanced-Level Training section). Role-play exercises are recorded or observed live by supervisors, who provide oral and written feedback, as well as numerical ratings on each interpersonal process skill  translating knowledge into coaching delivery and (2) applying interpersonal process skills to support engagement with digital CBT content. During these sessions, trainees participate in (1) brief digital CBT module content review, (2) intensive role-play exercises applying core process skills, and (3) introduction to protocols for managing advanced clinical issues .To continue supporting trainee development of interpersonal process skills and digital CBT content knowledge, trainees are continually rated on their process skills throughout intensive role-plays. Each week, supervisors review trainees’ intensive role-play segments and provide trainees with written feedback and numerical ratings on core interpersonal process skills. In addition, group supervision sessions incorporate oral feedback from supervisors and peer coaches, including in vivo corrective feedback during role-play exercises.Once trainees reach the advanced stage, the main goal is for trainees to achieve certification to serve as coaches for participants. This is accomplished by demonstrating (1) competency across all 6 core interpersonal process skills and (2) continued knowledge of digital CBT content. Advanced trainees conduct practice coaching sessions  with beginner trainees as mock participants. In addition to these practice sessions, advanced trainees attend a weekly supervision group consisting of intensive role-play exercises, with role-play targets focused on digital CBT content, interpersonal process skills, and management of advanced clinical issues .To support advanced coaches in progressing toward certification, advanced-level trainees receive written and numerical ratings on their full 30-minute practice coaching sessions. These ratings are used to certify trainees on competency across all process skills. Next, trainees achieve certification on digital CBT content by passing quizzes, which ensures knowledge of the intervention and promotes continued fidelity to the treatment model.Following successful advancement through the prior 3 stages of the program, trainees are certified to support the digital CBT with continued supervision. Certified trainees who are engaged in coaching continue to attend weekly supervision groups in which they discuss coaching sessions with their supervisor and peers. To ensure continued fidelity to coaching standards, supervisors review video recordings of each coaching session and rate the coaches’ application of process skills according to the behavioral rating scale described before. Video review further enables supervisors to use didactics and role-play exercises in response to common challenges or to address drift from the coaching protocol. Certified trainees additionally provide feedback to the supervision team to inform potential future iterations of the coaching program.Increased attention to trainee learning processes within mental health provider training and supervision procedures has potential to increase fidelity to EBTs . One wayvariability of learning contexts is applied through (1) independent trainee review of digital CBT content (outside of sessions), (2) didactic training (during sessions), (3) role-play exercises , and (4) participation in mock sessions (observed by the entire supervision group). Likewise, applying the principle of spaced instruction, digital CBT content and interpersonal process skills are introduced and revisited at multiple timepoints within and across training levels. Interleaved instruction is similarly used to promote initial learning of digital CBT content and process skills simultaneously . Furthermore, retrieval practices assess digital CBT knowledge throughout all stages of trainee development to support long-term retention of learning .From the outset of coach training, we have applied core principles of learning theory to guide the instruction of digital CBT content and process skills. For example, Following certification, ongoing fidelity-monitoring practices include (1) completion of a self-evaluation coaching checklist following all coaching sessions, (2) discussion of coach adherence to the digital CBT module during supervision, and (3) continued completion of mock coaching sessions during supervision with peer-to-peer and supervisor feedback.Following the acquisition of new knowledge and skills, competency-based supervision techniques can provide trainees with a pathway for transforming declarative knowledge into procedural knowledge -81. PrioIn this paper, we outlined 1 example of a scalable peer-to-peer mental health paraprofessional training and supervision program. Although many models of paraprofessional support have been described and tested previously, high demand and minimal resources have often corresponded with a reduced focus on fidelity monitoring and quality assurance . Lack ofWe believe this model has several notable strengths. Of note, our program focuses explicitly on fidelity, while also attending to the need for scalable care. As illustrated, the focus on fidelity is integrated into the program in 2 primary ways: digital technology as the primary agent for CBT content delivery  and contcore components, defined within the Consolidated Framework for Implementation Research (CFIR) as the “essential and indispensable elements” of the program, and the adaptable periphery, defined as the aspects of the program that can be modified and varied from site to site [Another strength is that our program is designed to be malleable and can be adapted in various ways based on implementation context factors. Along with fidelity, program flexibility is well established as a key ingredient to successful implementation of interventions in numerous settings ,85. Impl to site ,87. Incl to site . AlthougIn our program, we have identified several components of the adaptable periphery that have been tailored for various implementation contexts, with community partnership. For instance, although this paper describes implementation at 1 university, we are currently piloting coach training and supervision for the launch of STAND digital CBT in numerous other types of community settings, including local community colleges and health care systems. In partnership with community stakeholders, 1 example of a component in the adaptable periphery that we have modified to meet the needs of a new implementation site is the length of training time, which has been shortened to accommodate local resources. This has been accomplished by combining components of beginner and intermediate levels of training and including additional review and feedback of recorded role-plays outside of sessions to accelerate learning and growth. In another example of adaptation, we have worked with various sites to situate and design our coaching risk protocols  within the contexts of existing resources, infrastructure, and referrals. Another example of adaptation has been to integrate specific training on trauma-informed care strategies to support implementation of this program in communities with higher trauma prevalence rates. Cultural considerations are also essential, particularly in planning implementation of coach training programs in diverse settings such as ours. Working in partnership with community stakeholders to co-design cultural adaptations can lead to improved program acceptability and community engagement. Although we have made and discussed modifications within the adaptable periphery based on the unique implementation and contextual factors within various environments, the same guiding principles described in this paper serve as the foundational core components across settings.A final strength of our program is that it is intended not only to train students to serve as coaches to their peers but also to provide critical CBT skills to trainees themselves. Many coaches in our program anecdotally report that their experience throughout training has taught them invaluable interpersonal and cognitive-behavioral skills. In the broader literature, paraprofessionals describe feeling that their training experiences were associated with personal development and growth and increases in knowledge, self-confidence, and skill use . In the Several key limitations of our program should also be noted. First, because this program is situated within the scope of a large research initiative, ongoing funding has been available to sustain coach training and supervision. Beyond the realm of research, efforts to provide continuous funding for paraprofessional support programs in routine care settings are critical. In the initial iteration of our program, coaches have served as volunteers, engaged in all program elements as an additional responsibility outside of their other obligations. Data suggest that among volunteer staff supporting digital interventions, administrative issues, such as time constraints, may contribute to barriers to training completion and attrition . AdditioSecond, although we maintain a focus on fidelity in our program, the primary objective of our peer-to-peer program is to serve as a scalable model of care in real practice settings. Thus, given the resource constraints of real-world implementation contexts, we have designed our fidelity-monitoring procedures to minimize supervisor and trainee burden. However, in doing so, we recognize limitations in our capacity to optimally monitor fidelity, and acknowledge that fidelity is not monitored to the same degree in our program compared to standard clinical trials .Third, to maximize scalability of the program, coaching is provided virtually using videoconferencing. Prior research has raised the possibility that compared with self-administered or fully automatized options, digital mental health interventions may be most effective for adolescents and young adults when incorporating in-person elements . HoweverLast, our program was initially designed for use in a specific setting . Additional efforts and reliance on existing implementation science and human-centered design frameworks, such as the CFIR, are needed to determine how this program and similar ones may be adapted and augmented for use in other types of settings and with new populations. A number of conceptual frameworks to adapt interventions in new contexts have been proposed, and these can be used to guide adaptation of paraprofessional support programs for new settings .Finally, we consider future directions for this work, falling within the scope of the paraprofessional field at large. First, to meet rising rates of mental illness worldwide, expansion of paraprofessional mental health programs into new settings is critically needed. Second, funding for these programs must also encompass sufficient resources to support quality assurance in training, supervision, and treatment delivery , as has


The optimal cut-offs for tumor markers in CNS germ cell tumors (GCTs) to specify non-germinomatous GCTs are under active investigation. This study investigated 162 cases with histopathological confirmation and tumor markers, including 77 cases with robust specimens obtained from tumor resection. This study demonstrates that tumor markers are elevated in germinomas and teratomas, especially HCG in germinomas and AFP in immature teratomas, emphasizing the clinical significance of tissue diagnosis. Similarly, some biopsy cases without malignant findings on histopathology demonstrated tumor marker elevations, highlighting the limitations of reliable tissue diagnosis from a diminutive specimen. This study concludes that an integrated diagnosis based on tumor markers and histopathology is the key to precise diagnosis and, therefore, the avoidance of over- or under-treatment. This study also invites novel questions regarding whether marker-positive germinomas or teratomas should be treated as intensively as malignant NGGCTs, as well as the optimal treatment strategy for marker-negative immature teratomas.n = 162). Biopsy and surgical resection were performed in 85 and 77 cases, respectively. Among 77 resections, 35 demonstrated positivity for HCG, AFP, or both (45%). Seventeen of the marker-positive cases had no malignant non-germinomatous component identified on histopathology, but they were composed strictly of germinoma, teratoma, or both (49%). One embryonal carcinoma was the only marker-negative NGGCT in the study sample. Among 85 biopsies, 18 were marker positive (21%). Seven of these patients had no malignant non-germinomatous component on histopathology, suggesting the potential limitations of limited tissue sample volumes. Neither histopathological diagnosis nor tumor markers alone reliably diagnose NGGCTs due to the secretion of HCG and AFP by germinomas and teratomas. Treatment planning should incorporate integrated histopathological and laboratory-based diagnosis to optimize diagnostic and treatment strategies for this unusual and histologically heterogeneous tumor.The central nervous system germ cell tumor (CNS GCT) is a rare and incompletely understood disease. A major outstanding question in the 2015 consensus document for CNS GCT management was the utility and interpretation of the tumor markers human chorionic gonadotropin (HCG) and alpha fetoprotein (AFP) in the diagnosis of malignant non-germinomatous GCTs (hereafter NGGCTs) prior to treatment. In the current study, we assembled two geographically and ethnically different clinical cohorts from the Mayo Clinic (1988–2017) and the intracranial GCT Genome Analysis Consortium (iGCT Consortium) in Japan to address this question. Patients with both histopathological diagnosis and tumor markers available were eligible for inclusion ( Central nervous system germ cell tumors (CNS GCTs) are neoplasms, mainly occurring in pediatric, adolescent, and young adult populations, and they encompass several distinct histopathological entities. The most frequent histopathological variant is germinoma, which comprises more than half of the cases . Non-gerTumor markers (human chorionic gonadotropin (HCG) and alpha fetoprotein (AFP)) in serum and cerebrospinal fluid (CSF) are useful for diagnosis , the evaConceptually, this is motivated by the fact that most cases demonstrating elevated tumor markers are NGGCTs with a malignant component . However, it is generally accepted that treatment without histopathologic diagnosis exchanges procedural risk for a potentially non-indicated and toxic treatment, resulting in disagreement regarding the optimal tumor marker cut-off for initiating treatment without tissue diagnosis . This laAs GCT is a rare neoplasm that has a geographically distinct distribution worldwide, a straightforward investigation of the relationship between histopathology and tumor markers has not been conducted on a large international cohort. Furthermore, the contemporary moment is potentially optimal for a reassessment of current standard-of-care practices, given the widespread enthusiasm for minimizing any harm from treatment of trial enrollment, as well as individualization of care at diagnostic and therapeutic levels ,17,22,23With these considerations in mind, the primary goal of the current study was to investigate the critical and timely question of whether treating marker-positive GCTs as presumed NGGCTs with malignant components is reliable in a large, multicenter, international cohort study. Additionally, we assessed the ability of needle biopsy to adequately establish definitive diagnosis as compared to tumor markers or microsurgical resection.The present study is a retrospective cohort study of consecutive, neurosurgically managed, intracranial GCTs treated at the Mayo Clinic during the study period of 1988–2017. This yielded 80 primary cases . The histopathological diagnosis was performed at this institution. All pertinent aspects of the current study were approved and overseen by the institutional review board, including a consent waiver for a minimal-risk study.The intracranial GCT Genome Analysis Consortium (the iGCT Consortium) database was queried, and 154 primary intracranial GCTs were included for this study. These cases were registered from the neurosurgical departments in Japan participating in the iGCT Consortium. The central histopathological review, according to the WHO classification of tumors of the central nervous system, was performed by a single expert neuropathologist (Y.N.) . The invFrom these samples, all patients with histopathologic diagnosis and baseline tumor markers were included. Eligible tumor marker results included those measured in serum and/or cerebrospinal fluid (CSF), either during the preoperative period or intraoperatively. The resulting study cohort included 162 cases: 40 from the Mayo Clinic and 122 from the iGCT Consortium.There were 77 cases where either one or both of the tumor markers were available and surgical resection was performed . Serum and CSF HCG, and serum and CSF AFP are displayed as scatterplots across histology: germinoma (G), MT, ImT, G + MT, G + ImT, and NGGCTs containing malignant components  . This shImT was associated with modestly elevated HCG, although the values for all but one case were within the normal range. In contrast, most of the ImT in this cohort showed elevated AFP both in serum and CSF, similar to NGGCTs. This is reflected in the elevated AFP in mixed G + ImT cases.n = 18, 51%) were histopathologically verified as NGGCTs with a malignant component.There were 35 cases (45%) where tumor markers were above the diagnostic threshold (HCG > 100 IU/L or AFP > 10 ng/mL) and upfront resection was performed. The majority of these cases (Among the other 17 cases (49%), we observed 5 germinomas, 8 ImT, 1 germinoma with MT, and 3 germinomas with ImT . The detThere were 20 cases where tumor markers were negative and upfront resection was carried out. In 19 of 20 such cases (95%), no malignant non-germinomatous histological component was identified . One marIn total, 77 cases had at least one tumor marker and a surgical resection specimen larger than a biopsy, which were mapped in a tumor marker diagram . Most ofAmong 85 biopsy cases, 50 had data for both tumor markers. Within this subgroup, 11 cases (13%) had elevated tumor markers and malignant histopathology; 7 (8%) had negative histopathology in spite of elevated tumor markers; and 32 (38%) had negative tumor markers and negative histopathology. The seven mismatch cases included five germinomas, one germinoma with mature teratoma, and one mature with immature teratoma.We report the first large, multicenter, international study cohort assessing the utility of tumor markers when combined with histopathologic data—including a unique assessment of the relative diagnostic value of specimens obtained from biopsy as compared to those obtained from resection in establishing a reliable tissue diagnosis. Our data demonstrate compelling evidence that diagnostic accuracy is enhanced by combining histopathologic and laboratory data, as well as confirmation that the potential for sampling error in association with low-volume specimens from biopsy is a source of systematic error likely contributing to misdiagnosis. Correct diagnosis is needed to circumvent under-treatment resulting in the loss of a chance for a cure in NGGCT cases, as well as to avoid administrating toxic treatment to patients if they do not need it.This study demonstrates that germinomas and teratomas—especially ImT—are associated with elevated tumor markers in the absence of malignant histopathology. In this study, all histopathological specimens were carefully scrutinized, including a central review for cases registered via the iGCT Consortium, and an explicit review by an expert neuropathologist for cases diagnosed at the Mayo Clinic. Correspondingly, our assessment is that the probability of a multiply undiagnosed yolk sac tumor component among the ImT cases with AFP elevation is quite low. HCG elevation was seen in germinoma cases, while AFP elevation was observed in teratomas. HCG is well-known to be a reliable tumor marker for the detection of choriocarcinoma and choriocarcinoma components in mixed GCTs; however, germinoma has also been reported to express HCG at the RNA level, resulting in elevations of the tumor marker in serum and CSF ,25,26,27Treatment for ImT has been controversial. Marker-negative, histology-proven ImT is typically regarded as surgically treatable without adjuvant therapies in western countries; however, in Japan, consensus recommendations consider ImT as an intermediate prognostic group, prompting treatment with carboplatin and etoposide chemotherapy, as well as whole-ventricle irradiation. The latter strategy is based on their experience that resection alone or resection with limited irradiation results in a significantly increased risk of recurrence . AdditioAn important and fundamentally unanswered clinical question raised in the consensus document produced at the 4th International CNS GCT symposium in 2015 was the appropriate cut-off levels for tumor markers . The ideA particularly interesting outcome of the current study pertains to the biopsy cases, and the 7-of-50 subgroup in which no malignancy was noted on histopathology in spite of elevated tumor markers. Three of these cases had elevated HCG with germinoma histology, while four of these cases had elevated AFP—only one of which demonstrated ImT on histology (IMT). Given that elevated AFP in the setting of a pure germinoma is essentially unheard of, the more likely explanation of these unusual elevations in AFP are under-diagnosis, referable to the small sample volume obtained via needle biopsy. 20, should be investigated further to enrich the potential of both serum and CSF markers as more highly sensitive and specific diagnostic instruments in the future.The current study is subject to a range of limitations, including those pertinent to any such study of observational data. Our combined database is heterogeneous, consisting of cases from multiple institutions in Japan and a single US institution, with moderate variability in routine clinical practice and laboratory examinations between institutions. This represents a potential source of confounding or systematic error that could not be adjusted for using the available study data. Additionally, the study focused on a relatively narrow question of the relationship between histopathology and tumor markers, relegating other important clinical perspectives to future analyses. Finally, although the data cleaned from resection are clearly advantageous from a diagnostic perspective, this does not outweigh the additional morbidity of resection-first protocols, and so decision making in many circumstances will be unavoidably limited by the need for reliance on needle biopsy as the preferred method for tissue diagnosis, without a large tumor with symptomatic mass effect. Taken in concert with the study findings, these considerations emphasize the need for a careful and thoughtful interpretation of elevated tumor markers. Diagnostic alternatives, such as miRNAWe report a novel analysis of the relationship between tumor markers and histopathologic findings on either needle biopsy or surgical resection specimens. Our results concern a small but meaningful subpopulation of patients with positive tumor markers who did not have malignant GCT components on histopathology, indicating that some germinomas or teratomas are at increased risk of iatrogenesis if treatment proceeds without formal tissue diagnosis. As such, the question of whether marker-positive GCTs without malignant components in histopathology should be treated as intensively as other NGGCTs remains unanswered—in particular for HCG elevations, which appear to be more common than previously anticipated in non-malignant germinomatous GCTs. In tandem, we also identified cases of significant AFP elevations without malignant components, and we identified confirmatory histopathology on needle biopsy, highlighting the potential limitations of tumor under-sampling as another source of suboptimal treatment for potentially dangerous lesions. In summary, optimizing the diagnostic paradigm for GCTs remains an important area of study, and one that will almost certainly require utilization of both laboratory and pathologic data in order to maximize treatment benefit while minimizing unnecessary risk or harm to a population of young and vulnerable patients.


By extension, emerging infections include those caused by pathogens that are already present in the environment but previously did not cause infection or evolve a selective advantage for infection in a new host species2. Many factors may contribute to the emergence of a new zoonotic disease. These include evolutionary progression of virological determinants, changes in human demographics and behavior, dramatic increases in movement by people and animals, and environmental factors, such as ecologic and climatologic factors3.In the 21st century, newly identified pathogens, mostly zoonotic or vector-borne infectious agents, have consistently and relentlessly threatened public health and caused deadly outbreaks of global concern. “Emerging infectious disease” is a term used to describe previously unknown or known infectious diseases that have the potential to cause outbreaksIn this special issue, entitled “Emerging and Re-emerging Fatal Viral Diseases”, we focus on emerging RNA viruses, such as coronaviruses, as well as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus, and severe fever with thrombocytopenia syndrome virus (SFTSV); this issue will cover innate immune sensing and viral evasion strategies of coronaviruses, the evolution and future of influenza pandemic preparedness, and the latest findings regarding the emerging tick-borne SFTSV.4 summarizes the current knowledge about the mechanisms underlying host sensing and immune responses against coronavirus invasion, as well as the immune evasion strategies of coronaviruses.Humans are equipped with elaborate immune mechanisms to protect against harmful invading pathogens. The innate immune system constitutes the first line of defense against invading pathogens and is frequently targeted by immune-evading pathogens. Coronaviruses have evolved multiple strategies to evade host antiviral immunity. In hosts with immune naivety, distinct mechanisms associated with virulence, and pathogenicity include the perturbation of the host defense system by hindering antiviral innate immune responses through multiple viral inhibitory mechanisms. This results in successful viral transmission and adaptation to human hosts. Kasuga et al.5.Viral recognition by the host immune system results in the rapid production of type I and III interferons (IFNs), which are potent multifunctional cytokines that play an important role in host defense. Subsequently, IFNs and cytokines harmonize, resulting in a timely and balanced early immune response, which further activates host antiviral defense mechanisms by recruiting multiple immune cells to the site of viral infection. However, recent studies have reported that SARS-CoV-2 evades the host immune response by inducing a delayed interferon (IFN) response, which provides a window for uncontrolled viral replication. Inhibition and further dysregulation of type I and III IFN responses lead to hyperinflammation in coronavirus disease 2019 (COVID-19) patients. Moreover, it has been hypothesized that a delayed but exaggerated type I IFN response contributes to the severe progression of COVID-19. To further elucidate the immunosuppressive mechanism utilized by SARS-COV-2, Kim and Shin investigated the innate sensing mechanisms of this virus and the mechanism by which IFN inhibits SARS-CoV-2 replication6 and pose a perpetual global threat to humans. Due to continuous evolution, influenza has caused unpredictable and recurring pandemics for centuries. Since there is no way of predicting when and where a zoonotic pathogen will emerge, investigation at the first sign of emergence is particularly important. Thus, the World Health Organization established a global influenza surveillance network to monitor circulating influenza strains with pandemic potential in both humans and animals7. However, the occurrence of the 2009 H1N1 pandemic exposed a major weakness in the existing pandemic preparedness plan. Harrington et al.8 reviewed the history of influenza pandemic preparedness and proposed additional measures for the improvement of current influenza preparedness plans. In addition, they also address the intersection between the influenza pandemic preparedness network and the current SARS-CoV-2 crisis.Influenza viruses are emerging viruses belonging to the Orthomyxoviridae family that infect many species9 provided the latest findings regarding SFTSV, including information on vector and virus transmission, genotype diversity and epidemiology, a probable pathogenic mechanism, and clinical manifestations of human SFTS. In addition, the development of experimental animal models to further understand SFTSV pathogenesis is also discussed.Contributing to the emergence of zoonotic viral diseases is the capacity of viruses to adapt to diverse econiches, such as adaptation to and transmission by arthropods. A tick-borne infectious disease, SFTS, is caused by a novel phlebovirus and is an increasing public health concern. SFTS was first identified in China in 2009, and epidemics have occurred in several East Asian countries since then. With the increasing incidence of SFTS and the rapid, worldwide spread of SFTSV vectors, this virus clearly has pandemic potential and presents an impending threat to global public health. Casel et al.The emergence of novel fatal viral diseases that pose continuous threats and impose constant global health challenges has fueled intense efforts to improve the understanding of virus molecular and cellular biology. However, despite concerted efforts to elucidate the complexities of these emerging viruses, scant new information regarding the ever-changing threats they pose exists. This special issue provides a comprehensive review of and insights into current trending topics regarding emerging viral diseases. It is of invaluable significance to understand the intricacies of host–virus interactions, which is necessary to overcome emerging fatal viral diseases and to prepare for potential pandemics that may occur in the near future.