The first two microRNAs (miRNAs) were discovered in 1993 and 2000, but the scientific community uncovered their diversity only in 2001  cumulate two unusual features. They are composed of many related gene copies that tend to be repeated in tandem, and they are expressed only from one parental allele . These unusual miRNAs also share another commonality: their expression is markedly  specific to the placenta. While these features appear completely unconnected, their co-occurrence in three distinct genomic loci suggests a causal link: potential explanations are discussed in the Malnou et al. review.Novel miRNA genes have emerged throughout metazoan evolution, and this process is certainly still going on, generating taxon- or species-specific miRNAs besides the most highly conserved miRNAs. In their review article, Wightman et al. compiled a very exhaustive description of the known features of such miRNA degradation-inducing interactions.The regulation of miRNA degradation is still a largely open field of research. Recent discoveries showed that miRNA stability can be controlled by some of their target RNAs (either coding or non-coding). In that process, the main determinant for the induction of miRNA degradation seems to be the geometry of paired and unpaired nucleotides between the two RNA molecules. Examples of target-induced miRNA degradation are starting to accumulate, and in their review article, Girardi et al.'s review article.Among the biological pathways controlled by miRNAs in animals, sensitivity to viral infections constitutes an interesting case: while some miRNAs can play a clear antiviral role, some others are hijacked by viruses to promote viral infection. The complex interplay between mammalian viruses and miRNAs is described in Besides these four review articles, this special issue features three primary research articles, presenting novel results, and technological advances.Brustikova et al. identified chemical compounds with a reproductible activating or inhibiting effect. Carefully controlled experiments were used to assess reproducibility and estimate false positive rates, also revisiting previously published compounds.Using a high-throughput screen for small molecules affecting miRNA biogenesis or action, O'Neill et al. describe a novel computational pipeline for the annotation of small RNAs from Small RNA-Seq libraries. Applying it to actual Small RNA-Seq datasets from melanoma samples, they could identify and annotate small RNAs that are differentially expressed between treatment-sensitive and treatment-resistant cells.Bodak et al. convincingly show that miR-128 does not repress LINE-1 in murine ES cells, within good experimental detection limits. The clear effect of Dicer on LINE-1 expression in murine ES cells therefore does not rely on miR-128.An important issue in miRNA research pertains to the verification of predicted or published miRNA/target regulatory interactions. Because the human miR-128 miRNA had been shown to repress LINE-1 retrotransposons (Hamdorf et al., Overall, the seven articles published in this special issue provide a nice overview of open questions and established knowledge on several key questions in the field of miRNA biology. Technological and conceptual progress is very quick in this dynamical research topic: the presented articles are a great sample of the ongoing research in miRNA biology in animals.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Pancreatic adenocarcinoma remains one of the most aggressive cancers with an ongoing dismal survival rate despite some recent advances in treatment options. This is largely due to the typically late presentation and limited effective therapeutic options in advanced disease. There are numerous circulating biomarkers that have potential clinical application as tumour markers, including circulating tumour DNA (ctDNA), circulating tumour cells (CTCs), cell-free RNA (cfRNA), exosomes and circulating tumour proteins. This review will focus on the development of ctDNA as a non-invasive liquid biopsy, with its high sensitivity and specificity having potential clinical applications in pancreatic cancer. These include a role in screening, prognostication via the detection of minimal residual disease, early detection of recurrence, and for patients with advanced disease; tumour genotyping and monitoring treatment response. Prospective randomised adjuvant clinical trials are currently underway, exploring the impact of ctDNA-guided adjuvant therapy decisions. In this review, we provide perspectives on the current literature and considerations of future directions. Although not a common cancer, pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in developed countries and the seventh leading cause of cancer-related death worldwide ,2. PancrStudies utilising exome sequencing to assess clonal evolution of pancreatic cancer indicate a long window of opportunity may exist for early detection, maybe even in the realm of 20 years before any symptoms occur ,7. HowevCirculating cell-free deoxyribonucleic acid (cfDNA) was initially discovered by Mandel and Metais in 1948 , but it ctDNA has been restricted in its clinical application until recently due to the incapacity to reliably detect and compute the extremely low frequency of DNA variants that may be present in a blood sample, particularly in the context of screening of minimal residual disease. In an attempt to increase the sensitivity and specificity of ctDNA detection, specialised methods for analysis have been developed, ranging from technologies used for single mutation to entire genome analysis. However, ctDNA concentrations as low as <0.01% of the total cfDNA can now be detected, with ctDNA concentrations correlating with tumour burden ,16. The The lack of standardised guidelines on the use of ctDNA collection and analysis currently presents a clinical hurdle to overcome. As technology improves in assay development and costs decrease, the ability to reliably detect low frequency DNA variants in a blood sample will improve further, facilitating clinical realization.This review will focus on the evidence surrounding the clinical application of ctDNA in PDAC, given it is by far the most common form of pancreatic cancer. Further references to pancreatic cancer in this review will hence pertain to PDAC only.Diagnosing pancreatic cancer at an early stage is challenging as patients typically only have symptoms when more advanced disease is present and symptoms are often non-specific. However, in the absence of a good screening test, routine screening for asymptomatic individuals is not recommended . Even foCurrently, the only biomarker used routinely in the management of PDAC post diagnosis is carbohydrate antigen 19-9 (CA 19-9), a sialylated Lewis A blood group antigen . CA 19-9The diagnosis and staging of pancreatic cancer is dependent on imaging (computed tomography (CT), magnetic resonance imaging (MRI)), invasive endoscopic techniques  and exploratory laparoscopy. Multi-detector computed tomography (MDCT) is the first line imaging modality of choice for suspected pancreatic cancer, followed by MRI for equivocal cases or in the setting of a contraindication to CT . The senCurrent diagnostic techniques are far from adequate for early detection of pancreatic cancer. With the goal of developing improved strategies to diagnose pancreatic cancer at earlier stages, much focus has been placed on blood biomarkers including “liquid biopsies” such as ctDNA. Pancreatic cancer is an aggressive disease, but studies have shown there is an approximate 20-year window in which to detect pancreatic cancer before any symptoms occur ,7. This Bettegowda et al.  analysedIn an attempt to increase sensitivity, Cohen et al.  assessedThe uncertainty regarding the potential presence of KRAS mutations in benign conditions such as chronic pancreatitis should also be highlighted. Mulcahy et al.  detectedAs a standalone test, ctDNA currently remains restricted in its use in diagnosis due to the low sensitivity and the lack of prospective studies demonstrating the benefit of ctDNA-based screening, the most promising strategy being combining ctDNA measurement with protein markers to achieve greater sensitivity, potentially while maintaining a high specificity.Histologic confirmation is required to firmly establish a diagnosis of pancreatic cancer. However, in the context of high clinical suspicion, a non-diagnostic biopsy should not delay resection . Biopsy The molecular and genetic landscape of pancreatic cancer is innately complex and heterogeneous, with alterations in approximately 60 genes reported ,42. MostIn 2004, Uemura et al.  detectedTumour tissue sampling is currently limited by the need for repeat biopsies in the setting of false negatives, inadequate tumour sample, treatment monitoring, real-time assessment of metastatic disease, disease progression and evaluation of tumour heterogeneity. These limitations coupled with the inability to perform repeat biopsies considerably limits the use of personalized therapies in pancreatic cancer, a niche that liquid biopsies have the potential to fill.The management of early stage pancreatic cancer is evolving, with consideration of neoadjuvant therapy with either chemotherapy alone  or in cop = 0.03) and a longer OS (32.2 versus 19.3 months p = 0.027) than those who were ctDNA positive. Additionally, recurrence was detected 6.5 months earlier utilising serial ctDNA analysis compared with CT imaging alone  is currently active in Australasia; a multicentre randomised study that will enrol over 400 patients analysing ctDNA informed versus standard of care adjuvant chemotherapy in early stage pancreatic cancer. The study consists of two patient cohorts, with the major focus being on the cohort who received neo-adjuvant therapy prior to curative intent resection. Randomisation will occur after surgery. For those patients randomised to the biomarker-driven arm, ctDNA-negative patients will receive 3 months of adjuvant modified FOLFIRINOX. Those who are ctDNA positive, despite receiving pre-operative FOLFIRINOX therapy, will be switched over to receive 4–6 months of adjuvant gemcitabine-based doublet therapy. In a second separate cohort, this clinical trial will also investigate ctDNA guided adjuvant therapy in patients who undergo immediate resection.In the metastatic setting, there remain challenges associated with safely obtaining tissue for analysis, where liquid biopsies have a potential role. Concordance studies in pancreatic cancer have supported ctDNA-based tumour genotyping compared with tissue-based genotyping. The ability to noninvasively and reliably predict response to treatment as early as 2 weeks after initiation of therapy is a promising step forward and could become an integral part of patient management. Early information on response to treatment can guide clinicians in real time with regards to futility; avoiding the potential toxicity, inconvenience and cost of treatment with an ineffective therapy. An early switch to an alternative treatment gives the best chance of this being effective or potentially novel treatment concepts could be pursued. While currently not clinically relevant, the potential for ctDNA to detect tumour heterogeneity and the emergence of resistant clones under the selective pressure of a targeted therapy could also guide clinical decision making.Limited progress has been made in the diagnosis and management of PDAC. ctDNA analysis shows promise as a biomarker to be used from cancer screening through to the management of advanced disease. As always, this should be proven in prospective clinical trials, with a number of these already underway, with results eagerly awaited.


Older adults are more susceptible to adverse health outcomes during and after a disaster compared with their younger counterparts. Developing community resilience, or strengthening communities to reduce the negative impacts of disasters, has the potential support older adults’ health and well-being. Community-based organizations (CBOs), such as senior centers and Villages, provide social services and programming that support aging in place and may support older adults’ resilience to disasters. This study examines CBO leadership perspectives on the role of CBOs in building disaster resilience for older adults aging in place, as well as perceived barriers and facilitators to incorporating disaster resilience activities into organizational programming. In-depth interviews were conducted with a purposive sample of staff-members of CBOs serving older adults aging in place in King County, Washington. Participants included representatives from 14 organizations that varied in size, geographic setting, organizational structure, and ethnic, linguistic, and socio-economic backgrounds of organizational members. The sample included five government-run senior centers, seven non-profit senior centers, and two Villages. Interviews were audio-recorded and transcribed verbatim. We used a combined inductive and deductive approach to code and thematically analyze the data. Results indicate that local context, leadership risk perception, collaborations, and existing services and programming influence CBOs’ willingness to engage in activities supporting disaster resilience for older adults aging in place. Findings suggest that CBOs supporting aging in place may support disaster resilience for older adults by serving as a trusted source of disaster preparedness information and tailoring disaster-related messages for an older adult audience.


Genome-wide association studies have shed light on the association between natural genetic variation and cardiovascular traits. However, linking a cardiovascular trait associated locus to a candidate gene or set of candidate genes for prioritization for follow-up mechanistic studies is all but straightforward. Genomic technologies based on next-generation sequencing technology nowadays offer multiple opportunities to dissect gene regulatory networks underlying genetic cardiovascular trait associations, thereby aiding in the identification of candidate genes at unprecedented scale. RNA sequencing in particular becomes a powerful tool when combined with genotyping to identify loci that modulate transcript abundance, known as expression quantitative trait loci (eQTL), or loci modulating transcript splicing known as splicing quantitative trait loci (sQTL). Additionally, the allele-specific resolution of RNA-sequencing technology enables estimation of allelic imbalance, a state where the two alleles of a gene are expressed at a ratio differing from the expected 1:1 ratio. When multiple high-throughput approaches are combined with deep phenotyping in a single study, a comprehensive elucidation of the relationship between genotype and phenotype comes into view, an approach known as systems genetics. In this review, we cover key applications of systems genetics in the broad cardiovascular field. GWA studies have been conducted, largely in individuals of European descent, for sudden cardiac death  have demonstrated that chromatin interactions in human cells occur predominantly within domains with an average size of 880 Kb, known as topologically associated domains (TADs). These TADs are shared across a variety of cell types and are suggested to align with replication sites , or loci modulating transcript splicing known as splicing quantitative trait loci (sQTL). Additionally, the allele-specific resolution of RNA-seq technology enables estimation of allelic imbalance, a state where the two alleles of a gene are expressed at a ratio differing from the expected 1:1 ratio. Key mechanisms underlying allelic imbalance include the presence of truncating variants leading to nonsense-mediated decay of one of the alleles  analysis, identifying genetic variants modulating a specific quantitative trait. By subsequently overlapping the QTL with eQTL and sQTL data, either generated within the study or retrieved from public repositories (GTEx-Consortium cis inter-dependence), as well as the presence of epistatic effects . Bayesian approaches offer a means for true multi-variate statistical analysis that considers these aspects. Bayesian approaches for QTL analysis have been successfully applied to genetic association studies in animal models  genotype and gene-expression variance across individuals; (ii) epigenetic data, (iii) DNAse I hypersensitivity variance of SNPs and promoters, and (iv) expression variance of genes across multiple cell types; in addition to (v) LD blocks and (vi) imputed haplotypes inferred from the 1000 Genomes Project  rat strains  of single nucleotide polymorphisms (SNPs) for use in genetic mapping -driven inflammatory network enriched for viral response genes. The expression of genes in this network was shown to be regulated in multiple tissues by a single locus on chromosome 15. Subsequent analysis of the orthologous region and orthologous genes in human demonstrated association with type 1 diabetes for both, implicating the implicate Irf7 network genes and their regulatory locus in the pathogenesis of this complex cardiometabolic disease. Petretto et al.  are highly attractive for prioritization and identified multiple candidate genes as strong positional candidates for the investigated cardiometabolic traits. Lastly, Moreno-Moral et al.  pertaining to the HXB/BXH RI rat panel involved mapping genetic loci impacting on heart rate and ECG indices of cardiac conduction. QTL analysis on ECG parameters identified two QTL for PR-interval, respectively, on chromosomes 10 and 17. By further intersection of this data with cardiac eQTL data, we identified novel candidate genes associated with cardiac conduction. Subsequently expanding these findings by constructing co-expression networks enabled us to identify multiple gene networks associated with cardiac conduction, shown to be enriched for cardiac related processes through Gene Ontology enrichment analysis.One of our own recent efforts (cis associations between atrial transcripts and common genetic variants, their approach identified a causative gene for AF, MYOZ1.However powerful, translating genetic findings from animal models to human without functional validation is by nature limited through inference (Heinig et al. More recently, the study from Heinig et al.  reports Taken together, these studies suggest that combining genome-wide genotyping with transcript abundance data forms the powerful foundations of systems genetic approaches.Throughout the genetic studies discussed within this review, the loci and gene networks associated with the complex cardiovascular traits provide a starting point for future studies with the potential of identifying novel underlying mechanisms. The take-home message is that the main conclusion of all these studies could not have been reached without the applied integrative approaches: combining multiple measures of transcriptional control in a presence of a complex genetic background with multiple multi-variate analyses techniques.TTN, a gene for which many truncating variants have been identified that can lead to nonsense-mediated decay (Schafer et al. TTN, both the extent of the allelic imbalance and the number of known DCM genes affected varied greatly from patient to patient, yet all differential allelic imbalanced genes combined showed enrichment for DCM-related processes, as well as differential splicing and miRNA interference. This suggests a complex, heterogeneous genetic dosage effect across individuals, that in many cases will not be identifiable by standard GWAS, eQTL, and sQTL analyses. Additionally, this could suggest that such allelic imbalances are related to disease stage and may accumulate over time by interplay between genetic and epigenetic mechanisms, enabling early detection. Contrasting regular gene expression analysis, allelic imbalance can be reliably measured within a single individual, and several recent developments enable a statistically robust analysis (Knowles et al. Lastly, early detection combining molecular markers and clinical parameters is pivotal for preventive medicine in cardiology, but due to the heterogeneity of many diseases, this has proven to be exceptionally difficult. In the recent paper by Heinig et al. , by expl


Adipose tissue has been recognized as a complex organ with endocrine and metabolic roles. The excess of fat mass, as occurs during overweight and obesity states, alters the regulation of adipose tissue, contributing to the development of obesity-related disorders. In this regard, many epidemiological studies shown an association between obesity and numerous types of malignancies, comprising those linked to the endocrine system . Multiple factors may contribute to this phenomenon, such as hyperinsulinemia, dyslipidemia, oxidative stress, inflammation, abnormal adipokines secretion and metabolism. Among adipokines, growing interest has been placed in recent years on adiponectin (APN) and on its role in carcinogenesis. APN is secreted by adipose tissue and exerts both anti-inflammatory and anti-proliferative actions. It has been demonstrated that APN is drastically decreased in obese individuals and that it can play a crucial role in tumor growth. Although literature data on the impact of APN on carcinogenesis are sometimes conflicting, the most accredited hypothesis is that it has a protective action, preventing cancer development and progression. The aim of the present review is to summarize the currently available evidence on the involvement of APN and its signaling in the etiology of cancer, focusing on endocrine malignancies.  Obesity represents a condition of chronic excess fat mass. Several epidemiological studies have revealed an alarming increase in the number of obese individuals worldwide . It is iMolecular mechanisms linking excessive adiposity with the development of cancer are complex and still not completely known. Multiple factors potentially contribute to this relationship. Obesity is, in fact, often related to metabolic defects that may favor not only cancer initiation, but also its progression . These aIn this review we will summarize the currently available data on this topic, mainly focusing on endocrine malignancies , which seem to be deeply linked to dysfunctional APN secretion and action. APN is encoded by AdipoQ, a gene that makes a monomeric molecule made up of 244 amino acids and consists of a signal region at the NH2-terminus, a variable region, a collagenous domain and a globular domain at the COOH-terminus  18]. . 18]. After post-translational modifications, APN circulates in trimeric, hexameric, and multimeric high-molecular weight (HMW) isoforms. Each isoform is able to activate distinct signal transduction pathways, regulating various biological functions . MoreoveThe different APN isoforms mediate distinct effects in various tissues and organs. For example, the HMW isoform, which is the most biologically active, is believed to mediate the pro-inflammatory effects of APN, whereas the trimeric isoform has been suggested as responsible for its anti-inflammatory activity . APN acts through its classical receptors, AdipoR1 and AdipoR2, which have been demonstrated to have different binding affinity for the different APN isoforms . There aThe role of APN in endocrine cancer risk is deeply linked to many complex dysfunctions, including an altered adipose tissue homeostasis and the activation of multiple epigenetic pathways within tumor cells and neoplastic microenvironment . A correA robust amount of studies in the past two decades have suggested that APN exerts its antineoplastic effects on endocrine cancers via two main mechanisms. First, it can affect endocrine tumor growth by acting directly on cancer cells through receptor-mediated pathways. Secondly, it may indirectly influence cancer biology by modulating insulin sensitivity, inflammation and tumor angiogenesis . In the The main direct mechanism determining APN’s protective role on endocrine cancer cells is the activation of adenosine monophosphate-activated protein kinase (AMPK). This protein represents a crucial regulator of energy balance, as it is responsible for cellular adaptation to metabolically challenging states such as inflammation and oxidative stress. In such conditions, AMPK turns off anabolic and proliferative pathways, while increasing the production of adenosine triphosphate (ATP) . APN stiAPN also modulates signal transducer and activator of transcription 3 (STAT3) signaling. STAT3 is activated by adipokine-induced JAK phosphorylation and regulates various processes related to cancer development and progression . For exaOf note, APN influence in endocrine cancer cells may also depend on paracrine interactions between adipocytes and tumor cells, being that these cell types are in close proximity to each other . A typicThrough different mechanisms, APN can exert indirect antineoplastic actions, including insulin-sensitizing, immune-related and angiogenesis-related effects, although conflicting evidence has been published . An indiImmune system deregulation represents a crucial pathophysiological factor in determining increased cancer risk ,49. AbnoOne of the main mechanisms of cancer development is the recruitment of blood vessels to provide nutrients and oxygen to cancer cells . InhibitBreast cancer represents the most common malignancy in the female sex and the second most frequent tumor worldwide . In bothSeveral studies have investigated the role of adipocytokines in breast cancer, suggesting a pivotal role of APN in its development and recurrence ,64. WithAPN demonstrated in vitro anti-proliferative and pro-apoptotic effects on breast cancer cells, suppressing cell growth and proliferation, and inhibiting the migration and invasion capabilities of cancer cells ,67,68,69The mechanisms through which APN determines its protective role against breast cancer are yet unclear, but various molecular mechanisms have been proposed . SpecifiIt has been also suggested that APN’s effect on breast cancer growth may differ in relation to ERα expression. Most studies show that in ERα-negative breast cancer cells, APN has an anti-proliferative and pro-apoptotic effect ,90,91. IDespite the complexity of the association between APN and breast cancer, the preponderance of evidence suggests a correlation between low serum PN concentrations and breast cancer risk . FurtherEndometrial cancer is the most frequent gynecologic malignancy and the fourth most common type of cancer among women. It has a worldwide incidence of about 280,000 new cases annually .2) or severe obesity (BMI > 35 Kg/m2) have respectively a 2.5 times and 5 times higher risk of developing endometrial cancer than average-weight patients [Obesity represents one of the most important risk factors for the onset of endometrial cancer . An excepatients . This copatients . Moreovepatients .The excess of adipose tissue may exacerbate endometrial cancer risk through several other mechanisms, including insulin resistance, excessive aromatization of adrenal androgens in the adipocytes (resulting in higher levels of endogenous estrogens), chronic inflammation and the production of several adipokines (including APN) . In a reThe mechanisms by which APN inhibits the growth of endometrial cancer cells are not yet well known. However, several hypotheses have been formulated which imply the previously mentioned activation of AMPK (resulting in cell growth suppression and apoptosis), the extracellular signal-regulated protein kinase (ERK) and Akt pathway inhibition and the reduction of Cyclin D1 expression . FurtherNot only a lack of APN, but also a defect in its action seems to represent a negative prognostic factor that underlines the importance of this molecule in the prevention of the endometrial cancer . SeveralSummarizing, low circulating APN levels appear to be associated with an increased risk and worse prognosis of endometrial cancer . APN migOvarian cancer is the neoplasm burdened by the highest rate of lethality among female genital tract malignancies, and affects mainly peri- and postmenopausal women . The maiThe causes of ovarian cancer are not fully understood. The most important risk factors that have been identified are: genetic predisposition and history of ovarian neoplasia, Caucasian ethnicity, nulliparity, infertility, a high-fat diet and obesity .Little evidence is available on the role of APN in ovarian cancer risk and progression. Some authors analyzed serum levels of APN and leptin in 52 patients with ovarian cancer, showing that both APN and leptin concentrations were significantly lower in women with ovarian cancer than in healthy individuals . Other aFurthermore, Li et al. showed that AdipoR1 expression levels in cancerous ovarian tissues represents an independent prognostic factor of the disease, being positively associated with overall survival in patients . FinallyThyroid cancer is the most common endocrine malignancy . The majIn 2011, Mitsiades et al. showed that patients with any form of thyroid cancer had significantly lower levels of circulating APN compared to healthy subjects . These dSeveral in vitro studies have shown that even if thyroid cancer cells express both AdipoR1 and AdipoR2, papillary thyroid carcinoma cell lines express a significantly lower number of receptors than normal thyrocytes ,131. FinProstate cancer is the most common malignancy in males and represents the fifth leading cause of cancer death in men . A sedenIn contrast with these results, several authors have indicated that there is no significant association between APN expression and prostate malignancy , or thatSeveral genetic polymorphisms can result in a predisposition to increased prostate cancer risk. In a metanalysis of 133 published studies, AdipoQ rs2241766 and AdipoR1 rs10920531 variants were related to a higher risk of prostate neoplasia. Conversely, AdipoR1 rs2232853 variant was associated with a lower risk of developing this type of malignancy . FinallyIn conclusion, according to the predominance of literature showing an inverse correlation between APN and the risk of prostate neoplasia, APN deficiency might be a potential biomarker for the early detection of prostate cancer. Elevating APN levels in prostate cancer patients could be, therefore, a useful therapeutic target. Nevertheless, considering that literature data appear to be sometimes conflicting, further studies, both epidemiological and experimental, are warranted to clarify the association between APN and the development of prostate neoplasia.Metastasis is an extremely complex process that represents a major issue in the management of cancer. Besides its role in cancer promotion, aberrant APN secretion has also been associated with tumor spread and metastasis . RegardiAPN was also demonstrated to have a suppressive effect on metastatic endometrial cancer cells . APN wasFuture studies on the role of APN and other adipose tissue-secreting molecules on cancer invasion and metastatization should, therefore, focus also on LKB1-mediated effects, on the signaling pathways APN uses and on the potential interactions with other adipose tissue-secreting molecules that might contribute to the spreading of tumors.Since many studies associate endocrine cancer protection with APN-mediated signaling, drugs intended to bypass APN by directly activating its molecular pathways have been investigated in order to find potential strategies (both prophylactic and therapeutic) and counteract tumor development . HoweverFurthermore, it is also conceivable to increase endogenous levels of APN. Peroxisome proliferator-activated receptor-gamma (PPARγ) ligands have been proposed as a promising tool to reach this therapeutic target. It has been demonstrated that thiazolidinediones (synthetic PPARγ ligands) are able to increase APN concentrations in a dose- and time-dependent manner . Even ifObesity represents a major health and social problem strongly increasing the risk for various severe complications comprising endocrine cancer development. Our understanding on obesity-associated malignancies has been rapidly improving during recent years. Among other mechanisms, growing interest has been placed on the regulation of adipocyte-secreted molecules as a critical factor influencing cancer pathophysiology.APN has been recognized as a key mediator linking obesity and endocrine-related malignancies. The multifaceted role of APN includes a series of complex biological actions on different cancer metabolic pathways and tumor microenvironments. The majority of epidemiological evidence clearly demonstrates that hypoadiponectinemia is related to an increased risk of obesity-related malignancies and poor cancer prognosis. Nevertheless, APN has sometimes shown potentially contradicting actions in endocrine-related tumorigenesis. We believe that this topic represents a promising research field, but there remain several challenges before uniquely considering APN as a treatment strategy in cancer. A more profound understanding of the pathophysiological links between the different APN isoforms and endocrine-related malignancies is therefore required in order to develop effective and safe therapies. Moreover, molecular and environmental settings under which APN acts as an anti-inflammatory or pro-inflammatory adipokine need to be further examined. Finally, the specific roles of each APN receptor and signaling pathway on the different types of endocrine cancers also remain largely unknown. Further studies are therefore needed to clarify these aspects.


In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting  at the US National Cancer Institute in the summer of 2017. Here, we define ‘Microbial Therapy’ to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care. This White Paper is designed to increase the interactions between these growing fields of microbial oncolytics, and introduce their advances to the wider community of scientists and clinicians working on immunotherapies for cancer. We also describe the state of the field of microbial cancer therapy and point the direction where development and greater synergies with other fields are needed to increase the number of patients and indications that could benefit from this powerful modality.The fields of oncolytic virotherapy and bacterial therapy both exploit tumor-specific infectious microbes to treat cancer. As both disciplines have evolved into greater maturity, there has been an increasing appreciation that they share many features in common, to the point where a more consolidated approach  could be fruitful. For example, both modalities rely upon the ability of the therapeutic microbe to selectively infect and kill cancer cells Microbial therapy has had several prominent successes in recent years, including the commercialization of the first licensed virotherapeutic, Imlygic (T-VEC), and numerous viral and bacterial candidates progressing through clinical trials. The approval of Imlygic has heralded a renewed interest in microbial therapies. There are many common issues between virus- and bacteria-based therapies and addressing them concurrently will help advance and unify these two research communities. Specifically, we will spell out the major limitations still facing microbial therapies and what steps are necessary to promote greater translation into clinical use, especially in combination with other emerging modalities such as cancer immunotherapy.in situ production of cytolytic or immunostimulatory agents within tumor beds. Both viral and bacterial therapies have been shown to be capable of re-sensitizing tumors that have suppressed immune surveillance within the tumor microenvironment. Stimulation of the immune system amplifies the desired anti-tumor responses, clearing distant tumor tissue and preventing recrudescence of the cancer.Microbial therapies  are derived from naturally occurring microorganisms that have often been genetically modified to reduce systemic pathogenicity and increase anti-cancer efficacy. Microbial therapies eliminate malignant tissue by various mechanisms, such as The potential to cure cancer, or render some as chronically treatable, is the force that is driving discovery and innovation with microbial therapies. Microbial therapies have the potential to provide solutions to many clinical needs that cannot be addressed by current cancer therapeutics. These needs include treatment of refractory metastatic cancers, multidrug-resistant cancers, and cancers that evade immune clearance. This group affects almost all cancer sites including solid carcinomas of the digestive and reproductive systems, melanoma, sarcoma, and blood cancers. Due to the diversity and plasticity of candidate microbes, the development of multiple microbial therapies has the unique potential to address each of these widespread problems. Continual improvements in genetic manipulations have greatly sped up the time from idea generation and preclinical proof-of-concept to clinical testing, and these increases can only be expected to continue to improve in the future.An essential strength of microbial-based therapies is their specific targeting of cancerous cells and tissues. As such, microbial therapies are well-suited as therapies for metastatic disease, the primary cause of death from cancer. In addition, microbial therapies can circumvent the multi-drug resistant phenotypes that inevitably limit the long-term effectiveness of targeted small-molecule therapies. Microbial therapies have been created to reverse the immune suppression frequently exhibited in late stage tumors that is essential for the proliferation of many cancers ; and motMicrobiota can be manipulated, and these manipulations can be designed to offer features that may improve their ability to treat cancer. For example, the expression of microbial genes can be controlled by features of the environment, such as hypoxia, or other features of cancer cells, such as abnormal gene expression. Recombinant microbiota incorporating genetic features that are turned on or off in the presence of cancer-specific changes may enable a very large dose-response index, improving safety while enhancing the anticancer effects .Strategies for prevention with microbes is less developed than therapeutic strategies. Microbes that have been envisioned as cancer preventatives include prophylactic vaccines that target precancerous lesions and viruses that enhance and prime the immune response against precancerous cells. Microbial prophylactic vaccines have the potential to target cancers with viral etiology.Streptococcus pyogenes [S. pyogenes infection. He then began treating patients and had excellent results infecting cancer patients with S. pyogenes. His first patient recovered from head and neck cancer. Coley subsequently used killed S. pyogenes in combination with a second killed organism now known as Serratia marcescen bacteria [Microbial therapy against cancer has a long history. The interaction of bacteria and cancer has been known for several centuries. Records go back at least 200 years describing cancer patients going into remission after a bacterial infection . For exapyogenes . This stbacteria , 6. The bacteria , 6. JameHoption Cann et al.  comparedThe history of virotherapy for cancer is similar to bacterial therapy , 12. VirThe past few decades have seen an increase of systematic development and testing of microbial therapies. Enthusiasm for continued development of such therapies is due to early successes in preclinical and clinical studies. A number of viruses from diverse genera have been developed into anticancer therapies, several genera of bacteria have been tested in preclinical studies, and a few bacterial platforms have been tested in clinical settings , 19. TheAlthough initially designed as tumor lysing agents, the oncolytic virus (OV) platform also possesses activity as anti-vascular agents, gene therapy vectors, and their therapeutic capacity is intrinsically linked to their immune-stimulatory capabilities , 22. TheListeria monocytogenes designed to deliver recombinantly expressed tumor-specific antigens and the bacterium was well tolerated [For bacteria-based microbial therapies, many preclinical studies have shown retarded tumor growth and increased survival , 25. Comolerated –35.A prime example of a current cancer therapy based on an attenuated microbe is the treatment of superficial bladder cancer with the Bacillus Calmette-Guerin (BCG) vaccine. This modality likely functions by the nonspecific stimulation of immune responses against the tumor, and represents the only bacterial-based cancer therapy that is currently an established medical standard-of-care .Novel treatment strategies are urgently needed to fill the unmet medical needs for cancer patients where the four traditional categories of cancer management are not effective. These pillars of cancer therapy include surgery, radiation, chemotherapy and more recently, immunotherapy . Recent low and PD-L1neg, indicating poor response to ICB agents targeting the PD-1/PD-L1 axis. The poor efficacy of standard therapy for MPM  would be improved by the addition of new modalities. Unmet needs that could be addressed with microbial preventive agents include cancers with viral etiology such as hepatitis C virus (HCV), Merkel cell polyomavirus (MCV), Epstein-Barr virus (EBV), human herpesvirus 8 (HHV-8), and human T-cell lymphotropic virus type 1 (HTLV-1). While many other opportunities for microbial oncology therapeutics exist, they are most likely to get clinical traction in indications such as these examples where high-priority unmet needs exist. If durable clinical benefit is obtained in these indications, microbial-based therapies would be well positioned to address other oncology indications.Other examples of refractory cancers are melanoma, urothelial carcinoma, some lung cancers and malignant pleural mesothelioma (MPM). Two forms of melanoma that lack effective treatment are locally-advanced unresectable disease that is refractory to immune-checkpoint inhibitors, and refractory visceral metastatic disease. Similarly, in urothelial carcinoma, non-muscle invasive bladder cancer can be refractory to Bacillus Calmette-Guerin (BCG) and locally advanced muscle invasive and/or metastatic disease can be refractory to both platinum agents and ICB. In lung cancer, treatments are needed for patients who have failed chemotherapy and are PD-L1Microbial therapies have unique mechanisms to control cancer. It is the uniqueness of these mechanisms that provides the potential for these therapies to address unmet needs that cannot be attained with molecular therapies. There are three major mechanisms employed by microbial therapies to control cancer: gene delivery, immune stimulation, and direct oncolysis. For many viral therapies, these three mechanisms are intimately linked and are driven by single genes or single mechanisms. For bacteria, which are larger and contain more genetic material, these mechanisms can be integrated or built from disparate mechanisms.Most virus-based therapies are multi-mechanistic. These therapies function by delivering genes into the cancer host cells. Many of these delivered genes are designed to either directly engage the immune system or to lyse cancer cells. This lysis leads to the release of cancer specific antigens that in turn stimulate the acquired immune system. Virus therapies can be grouped into two types: cancer vaccines and virotherapies. Vaccines encode and express cancer-specific antigens or epitopes. In contrast, virotherapies do not encode exogenous tumor antigens but drive lytic mechanisms that kill cells to unmask or unleash endogenous antigens from the cells that become infected. “Oncolytic vaccines” are a hybrid strategy, in which an oncolytic vector also encodes a tumor antigen or neo-epitope.Viral therapies can utilize either replicating or non-replicating viruses. Replication-competent viruses retain the ability to replicate and spread to adjacent un-infected tumor cells and are, in theory, dose amplifying. Eventual death of virus-infected tumor cells ensues a second mode of efficacy resulting from host anti-tumor immune responses roused by the release of tumor antigens along with inflammation due to infection. Together this has the potential to educate a host memory response to clear both injected lesions as well as un-infected metastatic disease.Most nonreplicating viruses are utilized predominantly as gene delivery vehicles that can deliver toxins, tumor suppressors, genes that result in drug sensitization to chemotherapy, or genes that convert a non-toxic drug into its toxic intermediate . These aMany viral strains have been exploited for anti-cancer drug development. DNA viruses include: Adenoviridae, HSV-1, Parvoviridae (see Case Box #2), and Poxviridae. RNA viruses exploited for development into tumor agents of destruction include Paramyxoviridae, Picornaviridae, Reoviridae, Retroviridae, and Rhabdoviridae . Some stViral vectors are the default selection for gene-delivery therapies. Research over the past three decades has led to a deep understanding of viral tropism, genome sequences, regulatory elements and immune-evasion mechanisms. These efforts have culminated in the development of a versatile arsenal of increasingly tumor-selective viral-vector technologies. Replacement of naturally-occurring viral genetic-regulatory elements with tumor-specific elements has been a key enabling feature that has led to the generation of numerous tumor-selective viral vectors , 61. TheTo enhance the immune response, the processing of specific antigens can be altered. For example, gene sequences encoding fusion proteins linked to intracellular-trafficking elements have been demonstrated to improve and enhance antigen presentation , 64. Thein situ vaccination, such as developing a plant virus, cowpea mosaic viral-like nanoparticle, as a potent ISV reagent [Another approach to cancer vaccination is “in situ” vaccination (ISV). Vaccines exploit antigens to be recognized by the immune system and adjuvants to activate the immune system, but for cancer, the problematic issue has been to choose exactly which antigens are the “right” ones. ISV uses the tumor as the source of the antigen and injects the adjuvant directly into the tumor. The overall goal is to generate a strong local antitumor response that reverses the local tumor-mediated immunosuppression and then to have that local response become systemic to fight potential or recognized metastatic disease , 68. For reagent .in situ “oncolytic vaccination”, at least partly promoted by antigen cross-presentation [One limitation of cancer vaccines is that, although tumor-associated antigens have been well characterized, the presence of tumor-specific neoantigens generated by somatic mutations within the genome of a cancer cell, are most commonly private, with shared antigens representing a very small percentage of known antigens . Consequentation . To bettentation . An alteentation . There aOne advantage of recombinant viruses that are oncolytic, is that they are capable of causing tumor cytoreduction, but also capable to elicit adaptive immune responses to the antigens, including neoantigens released by tissue injury and death induced by the viral life cycle. Immune activation by oncolytic-virus infection of tumor tissue comprises both, immediate effects of innate immunity and also adaptive responses for long-term tumor-specific activity. Much of the enthusiasm surrounding oncolytic vaccines revolves around their appeal as multi-mechanism therapeutics . Oncolytin vivo to break peripheral tolerance of tumor-antigen-specific effector cells. Measurable clinical responses to microbial therapies designed for this purpose have been achieved and, while response rates are low, they are significant, appear to be durable, and highlight the promise of this approach.This approach to microbial-based cancer therapy may be particularly well suited for use in indications not considered to be highly immunogenic, but where overexpression of specific tumor-associated antigens could be of value. These recombinant microbes are engineered to deliver or express tumor associated antigens in professional and immature antigen-presenting cells Similar to viral therapies, bacterial therapies can function predominantly by direct oncolysis or by engaging the immune system. Direct oncolysis can be mediated by secreting exotoxins or competing for nutrients . AlternaAn essential strength of bacterial therapies is their specific targeting of cancerous cells and tissues. Each modality targets in a distinct way. For example, active bacteria have been engineered to only colonize the microenvironment of tumors , 80; oncListeria monocytogenes (Case Box #3), Clostridium novyi (Case Box #4), and Salmonella largely because our understanding of their pathogenicity, physiology, and genetics has led to well defined attenuation strategies critical for safe use of live bacteria in patients. Each genus of bacteria is better suited to a specific mechanism. The predominant organism for immune engagement is Listeria because of its propensity to invade immune cells. Facultative enterics, like Salmonella and Escherichia, have been used for both purposes because of their ability to stimulate the innate immune system and because of their genetic plasticity. Obligate anaerobes, such as Clostridium, can localize in hypoxic regions of tumors, a characteristic that could circumvent adverse features associated with resistance to radiation therapy or chemotherapy [A large number of bacterial genera have been investigated as potential therapeutics Table . The bacotherapy .Table 3BListeria monocytogenes, Salmonella enterica, and Clostridium novyi-NT. All of these bacteria selectively survive and multiply in the TME. For example, attenuated Listeria monocytogenes have been designed to deliver recombinantly-expressed tumor-specific antigens [Salmonella enterica have been engineered to secrete biologically-active cytokines such as IL-2 [The intrinsic ability to stimulate the innate immune system makes bacterial-based cancer vaccination strategies an attractive approach. Many genera of bacteria accumulate in the tumor microenvironment and once there can convert the tumor microenvironment (TME) through proinflammatory responses. Tumor-colonizing bacteria alter the immune suppressive environment of tumors and make them immune stimulating. This conversion can be achieved through different approaches, such as (1) incorporation or cloning of adjuvants into the bacteria, (2) cloning highly immunogenic antigens as an alternative for neo-antigens, (3) encoding with immunogenic cytokines which evoke anti-tumor immune responses, (4) cloning of checkpoint inhibitors into the bacteria, or (5) depletion of immune suppressing macrophages or myeloid-derived suppressor cells (MDCS). Species that have been investigated for this approach include attenuated antigens . Similar as IL-2 , 85.Many classes of microbial anticancer therapies rely upon induction of immunogenic cell death (ICD) to propagate effective anti-tumor immunity. ICD is recognized as a critical process that initiates the tumor-immunity cycle and ultimately leads to an adaptive immune response . In cont+ T cells are a major player in the adaptive antitumor immune response [+ T cells to low-density tumor antigens by enhancing proximal T-cell receptor (TCR) signaling [Bacterial-based vectors naturally stimulate the innate system through activation of multiple immune pathways. Diversity among membrane components between bacterial species manifests in differential expression and presence of structurally-conserved pathogen-associated molecular patterns (PAMPs). These molecules include flagella, pili, adhesins, lipotechoic acid and lipopolysaccharide and each activates specific toll-like-receptor (TLR) family members to elicit distinct innate immune-signaling cascades that ultimately translate to a comprehensive immune signature unique to each bacterial organism . Differeresponse , 91. A bignaling .In addition to their intrinsic anti-cancer effects, bacterial vectors can be genetically engineered or modified to express proteins with direct tumoricidal properties . BecauseEscherichia coli [Staphylococcus aureus [The most prominent bacterial toxins exploited as therapeutics are pore-forming cytolysins derived from hia coli , 100, ors aureus , 102. Ths aureus . They ars aureus –107. Sevs aureus , 108–111s aureus  and are s aureus , 108. Ass aureus , 112. HoE. coli has been transformed to transcribe shRNAs from a plasmid containing the invasin gene inv and the listeriolysin O gene hlyA. These two genes encode bacterial factors that are needed for transfer of shRNAs into tumor xenografts [Immune-cell-targeting bacteria, such as Listeria monocytogenes, can reduce tumor mass by delivering radioactivity. For example, 188-rhenium has been coupled to Listeria through anti-Listeria antibodies and shown to be effective against mice with pancreatic cancer , 114. Banografts .The greatest therapeutic strides with oncolytic platforms are likely to be achieved for diverse patients through multipronged pharmacological and immunomodulatory approaches. One approach is to identify chemotherapy drugs that can alter cellular signaling to sensitize tumor cells to viral replication and/or burst. Cellular DNA damage, after induction by radiation or chemotherapy, can augment HSV-1 replication by amplifying viral burst . Based oThe greatest attention has been paid to combination with checkpoint-inhibitory antibodies –124. TheWhile many bacterial-based therapeutics impart significant single agent activity, combination with other modalities may increase efficacy. Bacterial proliferation is often specific to the immune-privileged necrotic/hypoxic regions of solid tumors , 131, spNon-living bacteria-based therapies are alternative strategies that have several advantages over living organisms. This category of therapies includes bacterial minicells, cell-wall complexes, outer-membrane vesicles, and other bacterial derived therapies –139. BacKlebsiella), stimulates a lung-specific anti-cancer immune response, including macrophage recruitment and M1 polarization, NK cell recruitment and activation, and upregulation of the NKG2D pathway [Site-specific immunomodutors (SSIs) are composed of components of specific bacterial pathogens. When administered subcutaneously, SSIs result in a recruitment and activation of innate immune cells (including macrophages and NK cells) in the organ in which the bacterial pathogens, from which they are derived, commonly causes infection. For example, QBKPN, derived from a lung pathogen . For example, experience with cancer vaccines has demonstrated that targeting tumor-specific antigens alone is likely insufficient to generate effective anti-tumor immunity and to prevent immune escape. Optimal T-cell responses come from not only the engagement of the T-cell receptor binding to the MHC-peptide complex  but also to a second signal with cytokine stimulation or a costimulatory molecule . Two FDA-approved cancer vaccines highlight this concept. Sipuleucel-T (Provenge), an autologous dendritic cell vaccine contains a fusion protein (PA2024) consisting of the target antigen (prostatic acid phosphatase) as well as immunostimulatory GM-CSF, which can augment dendritic cell activity and maturation. Similarly, T-VEC produces GM-CSF to enhance immunogenicity .An intriguing story has emerged suggesting that effective cancer immunotherapy can promote “antigen spread” . The notNumerous studies investigating the role of innate anti-viral defense responses in ocolytic viral therapy have uncovered that rapid virus clearance minimizes the initial burst of tumor cells and thus limits virotherapy by negatively regulating virus lytic tumor destruction . While iResults from preclinical and clinical studies suggest that tumor-targeting bacteria may be a useful option for cancer treatment (see Case Box #6). However, toxicity will be a major challenge for their clinical development. A successful outcome may be achieved only when toxicities associated with the therapeutic infection can be effectively managed without compromising the therapeutic effect. Despite the complex safety considerations, the FDA has allowed several clinical trials with tumor-targeting bacteria and bacterial vaccines designed to elicit a systemic response Table  10, 149149–152. Because of the potential toxicity of these therapies, knowing when and how to intervene are critical issues which will have direct impact on clinical outcomes. Dose determination is another important issue with the clinical development of bacterial therapy. Dose escalation is a standard practice in Phase I clinical trials. However, the effective dose for live bacteria depends more on the target tumor tissue where they can multiply than on the administered dose. A large tumor with extensive necrosis and minimal infiltration of inflammatory cells is more likely to support a robust bacterial infection, resulting in pronounced tumor response and toxicity, regardless of the administered dose. In contrast, a small tumor or a tumor with minimal hypoxic/necrotic regions is unlikely to show significant response even if a large number of bacteria are administered.To fully capture the potential of microbial therapy it would be beneficial to describe the design space as comprehensively as possible. An optimal microorganism must (1) target only tumor cells, (2) have tolerable toxicities or side effects, and (3) be generically stable. These organisms would act by (1) engaging the immune system, (2) reducing tumor-associated immunosuppression, or (3) delivering anticancer agents . Targeting could be enhanced by generating microorganisms that (1) selectively infect poorly vascularized tissues not accessible to drugs; (2) specifically target precancerous cells, metastatic tumors, and/or dormant cancer cells; (3) target tumor neovascularization; or (4) have organ-specific affinity (e.g. respiratory viruses). Microbial cancer therapy could be effective beyond direct treatment as (1) preventatives and vaccines, or (2) by preventing tumor recurrence.What mechanisms control (1) microbial tropism to tumors and metastases, (2) invasion and microbe-mediated lysis of tumor cells, (3) microbe-initiated immune responses, and (4) processing and presentation of microbe-delivered or tumor-cell derived antigens?How can toxicities and deleterious side effects be managed, eliminated or prevented?What mechanisms can be introduced into bacteria and viruses to increase genetic stability?Can the immune system be selectively evaded to limit microbial clearance and inactivation?Can new targets be identified for microbial recognition of tumor cells?What is the optimal balance between microbe-initiated immune stimulation, microbial attenuation, and immune evasion?Are there cancer types particularly sensitive or resistant to microbial therapies? What biological characteristics make a tumor more sensitive or resistant to a microbial therapy?Can microorganisms prevent the transformation of precancerous cells into tumor cells?Can microorganisms eliminate residual cancer cells after treatment?Can companion diagnostics be used to predict the response to microbial therapeutics?To achieve these goals, many questions must be addressed about the interactions of the microbes with tumors and tumor cells. Each of the following questions is a furtive research area that, when addressed, will be instrumental in the development of effective microbial anticancer therapies.Most of the questions about the mechanisms of microbial therapies can be classified into four categories: simulation of the immune system, control of toxicity, control of delivery, and safety.To date, the complex effects of a viral tumor infection on the TME and the consequences for the tumor-infiltrating immune cell compartment are poorly understood. There is growing evidence that a tumor infection by an oncolytic virus opens up a number of options for further immunomodulating interventions such as systemic chemotherapy, generic immunostimulating strategies, dendritic cell-based vaccines, and antigenic libraries to further support clinical efficacy of oncolytic virotherapy . More sp+/CD8α+ dendritic cells (DCs) are critical for cross-presenting tumor antigens in tumor draining lymph nodes (TDLNs). Cross presentation allows for priming and expansion of naïve CD8+ T cells that recognize the cognate antigen [+/CD8α+ DCs include systemic administration of FMS-like tyrosine kinase 3 ligand (FLT3L) or intratumoral delivery of immune adjuvants such as TLR3, TLR7, and STING agonists.Novel approaches are needed to alter the immunosuppressive microenvironment of tumors and facilitate the recognition of tumor antigens. The lack of immune-cell infiltrates within tumors negatively correlates with the response to immunogenic cell death (ICB) and patient survival , 158. Im antigen , 160–162Bacterial infections of tumors naturally stimulate an immune response. However, it is not entirely clear how intratumoral infections enhance antitumor immune responses. It has been suggested that the antitumor immune responses result from the infection-induced immunogenic immune-cell death causing epitope spreading . A complOne of the major questions affecting bacterial therapy is how to produce a high enough local concentration of a therapeutic. Compared to viruses, bacteria are larger and can be used to deliver multiple gene products. However, bacteria are small relative to mammalian cells (about 1000-fold by volume), meaning that any produced protein must be effective at low concentrations. Regardless of the mode of action, efficacy depends on the local concentration of the therapeutic molecule. A high local titer can be achieved by manipulation of bacterial gene expression, metabolism, or protein secretion. The local tumor concentration could also be enhanced by increasing tumor colonization and proliferation. Better understanding of these mechanisms could greatly improve bacterial therapy.In comparison, viral therapeutics have limited coding capacity due to small genomes and are limited to delivering only a small number of genes. In the context of delivering an immunomodulatory gene sequence, most viral vectors deliver a single gene  and conflicting data raise questions as to whether this is sufficient for immune activation robust enough to break peripheral tolerance to tumor antigens. Strategies to address this may include addition of other immunomodulators (e.g. TLR agonists such as imiquimod) to therapeutic regimens, and/or use of viral vectors encoding regulatory RNA sequences implicated in the global regulation of immunosuppression or pathways at the level of the transcriptome.The control of localization and delivery is essential for bacterial therapies. Focusing therapy specifically to malignant tissue can greatly increase efficacy and reduce toxicity to normal tissue. Early clinical trials with bacteria showed that facultative anaerobes colonize human tumors, but at lower densities and with more heterogeneity than in mouse models . AlthougThere are three categories of bacterial therapies based on how they accumulate in tumors: obligate anaerobes (e.g. Clostridium and Bifidobacterium), facultative anaerobes ; and immune-cell-targeting bacteria (e.g. Listeria). The spores of obligate anaerobes germinate exclusively within the hypoxic regions of the cancer , 26, 164Because viral particles are inert outside of host cells, classic delivery of naked virus depends on passive accumulation. High viral titers can be safely administered when using a highly tumor-selective virus and tumors with leaky vasculature. To be effective, the inoculum needs to remain in the circulation for sufficient time to allow passage through the tumor vascular bed. This becomes a particular issue for viral species that have not evolved to spread in the blood, and so have little or no protection against complement or removal by the reticuloendothelial system , 176. EvThe easiest way to avoid such delivery hurdles is to directly inject the virus into the tumor. Intratumoral administration would circumvent antiviral immune responses, enable more direct contact with cancer cells and reduce systemic toxicity . HoweverSeveral strategies have been developed to try to enhance the systemic delivery of oncolytic viral therapies. One approach has been to chemically modify the viral particle itself , such asex vivo cells that are known to traffic to the sites of tumor beds following infusion into the patient. In this way, cells (known as “carrier cells” or “Trojan horses”), including some stem cells or immune cell populations that naturally traffic to and infiltrate tumors, can be used as delivery vehicles to actively carry the viral payload to the tumor target [in situ carrier vehicles to enhance spread within a host. These phenomena have also been shown to occur in the clinic resulting in enhanced delivery of oncolytic viruses, such as reovirus [Another strategy that has demonstrated success in pre-clinical models is to pre-infect r target . This hareovirus . As suchin situ vaccination has primed a systemic immune response . It remains unclear if systemic administration can improve response rates without additive or synergistic toxicity. Because human bodies have evolved to fight infections, immunity to microbial agents may develop in response to systemic administration. Major challenges in the development of systemic delivery are serum neutralization and hepatotoxicity. A neutralizing antibody response has been observed in almost every patient treated with viruses, but has not been correlated with a response or lack thereof.Modern microbial therapies have an excellent safety record. In almost all oncolytic virus trials to date, few adverse events have been attributed to replicating viruses. However, off-target effects and viral mutation/transmission remain ongoing concerns. An open question is the optimal administration of oncolytic viruses. When replicating viruses are administered systemically, non-malignant cells are spared because they retain more robust anti-viral defenses than transformed cells. Infected normal cells either destroy the oncolytic virus or commit apoptotic suicide resulting in a self-limiting infection. As demonstrated by intralesional administration of T-VEC, distant tumor regression suggests that Numerous tumor-targeting bacterial strains have shown a therapeutic benefit in experimental tumor models for decades, and yet clinical development has been slow to follow. Safety is a major concern for therapy with live bacteria, where therapeutic effect is intertwined with toxicity. Preclinical pharmacologic and toxicologic studies with live bacteria have shown satisfactory safety profiles in both healthy and tumor-bearing experimental animals , 29. HowThree issues that are essential for the development of microbial therapies are 1) finding the most appropriate predictive animal models, 2) development microbial therapies into cancer prophylactics, and 3) defining standard techniques for GMP manufacture.Animal models are invaluable to the understanding of neoplastic development and to the evaluation of novel therapeutics. Each model has its benefits and deficiencies which should be carefully considered prior to the study. The implementation of multiple models over the course of the translational development path will aid in recapitulating as many aspects of human biology as possible. The choice of model should balance cost and efficiency with accurate representation of pathology and progression.Allo- and xenotransplant models provide time-saving experimental protocols and less expensive animals than more complicated models. The use of immunocompromised xenotransplant models can provide information about the efficacy of oncolytic therapies, but are inappropriate for immunotherapies. Immune-deficient mice lack the ability to develop an adaptive memory response against both the microbe and the implanted tumor. Although allo-transplant models are immunocompetent, the speed of tumor growth in transplant models can prevent development of systemic or TME immune states present in human cancer .Genetically engineered mouse models (GEMMs) may more acurately recapitulate essential aspects of human cancer . AutochtPatient-derived xenografts (PDX), reconstitute the heterogeneity of the genomic aberrations seen in human cancers. This property overcomes the major limitation of GEMMs, but introduces additional limitations. In this model, primary human tumor tissue is introduced into “humanized” mice that have been reconstituted with a human hematopoietic system . This moThe use of rodents to determine the safety and efficacy of novel agents has inherent limitations because infectious biological agents have co-evolved with mammals and are species-specific pathogens. The inherent resistance of mice to some human pathogens makes preclinical testing of these microbial agents particularly challenging. Alternate models to evaluate the safety and/or efficacy are Syrian hamsters and non-human primates.Evaluating therapeutic efficacy in veterinary clinical trials is also a useful approach as it permits safety and efficacy testing in large spontaneous tumor models –202. SpoMicrobial therapies have the potential to be potent cancer prophylactics. To be effective, prophylactic vaccines could (1) target cancers with viral etiology, (2) directly target precancerous lesions, or (3) enhance the immune response against precancerous cells. Some cancers have a viral etiology including liver cancer, cervical, head and neck, anal cancer, Burkitt Lymphoma, nasopharyngeal carcinoma, Merkel Cell Carcinoma, Kaposi Sarcoma, and HTLV-1 associated T-Cell Leukemia/Lymphoma. While the effectiveness of vaccination against cancers with viral etiology has been demonstrated for human papillomavirus (HPV) and hepatitis B (HBV), other vaccines are needed to target additional viruses that are highly associated with cancer, including HCV, MCV, EBV, HHV-8, and HTLV-1. Microbial autologous DNA/RNA vaccines could prevent recurrence by targeting known tumor-associated antigens. Targeting precancerous lesions would prevent the transformation into tumor cells. These vaccines could be prepared to target antigens from patient-derived tumors or human tumor cell lines. Prophylactic vaccines might be useful in cases where there is known family history risk, including known genetic determinants that lead to a high risk of cancer. Microbial-based therapies could also prevent cancer by eliminating cancer-associated microorganisms, or by targeting neovascularization and dormant cancer cells. Microorganisms that target tumor antigens would also be useful for eliminating residual cancer cells after treatment.The GMP manufacture of microbial-based cell therapies is a regulated process that centers on product safety, consistency, and stability. Any investigational new drug (IND) application submitted to the United States Food and Drug Administration (FDA) is required to have a comprehensive description of the chemistry, manufacturing, and control (CMC) information used while generating the product. The FDA has recently published detailed information, in the form of an industry guidance, on the information that must be submitted in an IND pertaining to bacterial- or viral-based biologic products .Critical to the CMC package for any biological product manufactured under GMP conditions is the ability to properly characterize the sterility, purity, identity, potency, and stability of the drug substance and drug product, including its raw materials, and critical intermediates. GMP manufacture of microbial-based cell therapies begins with raw materials and a list of each component used in the manufacturing process. Second to raw materials is the generation of a seed stock, expansion and characterization of the microbial Master Cell Bank (MCB), and any further expansion into a working cell bank (WCB). Key considerations in the manufacture of the seed stock, MCB and WCB include verification of the absence of any lysogenic prophage (for bacteria), genome sequence information characterizing any chromosomal modifications (deletions and additions), phenotypic confirmation of attenuation, microbial purity , cell viability, and the stability, restriction digest pattern(s), and sequence identity of any episomal constructs . The stability of these features within the MCB and WCB should be characterized over time to gain an understanding of when failure occurs. During early phase production, a master manufacturing record (MMR) is established based on at least one process development experience at the intended manufacturing scale. The MMR is subject to industry standard Quality Assurance (QA) and Quality Control (QC) review procedures and sponsor approval before being accepted for use and employed to officially codify and record manufacturing steps.Manufacturing processes are typically divided into upstream, downstream, fill/finish, and release testing components. For bacterial cell therapies, the upstream process usually begins with MCB/WCB vial thaw and includes fermentation and formation of the crude cell paste. Downstream processing of bacterial cell therapies terminates after formulation and that process intermediate is defined as the formulated drug substance. A suite of release tests is typically performed on the formulated drug substance prior to advancing to the fill/finish stage. In fill/finish, the drug substance is individually vialed, packaged, labeled as appropriate and is now defined as the drug product. Vialed drug product is subject to further release testing and released for stability testing and use in clinical trials. For non-living bacteria-based products and virotherapies, sterility must also be determined prior to use. With living bacterial cell therapies, demonstrable microbial purity is required.The National Cancer Institute (NCI) offers translational research assistance for microbial-based cancer therapies through programs such as the NCI Experimental Therapeutics (NExT) program Table . The NExhttps://frederick.cancer.gov/Science/Bdp/Default.aspx). Over the past 15 years the BDP has provided NCI with expertise in developing biologic products such as recombinant proteins, monoclonal antibodies, immunotoxins, cell banks, immunocytokines, vaccines, and mammalian viruses. BDP technical expertise includes bacterial fermentation, mammalian cell culture, protein and virus production and purification, and aseptic fill/finish manufacturing operations. Analytical capabilities include protein biochemistry, bioanalytical testing, physio-chemical analysis, virus testing, molecular biology analysis, stability assessments, and standard quality control techniques for raw materials and facility operations. The BDP manufacturing capabilities and regulatory support are most appropriate for IND-enabling and clinical use. Once a commercial partner is identified, BDP transfers the technology to the company for commercial manufacturing and licensing.The Frederick National Laboratory for Cancer Research’s (FNLCR) Biopharmaceutical Development Program (BDP) is one such contractor used for development of clinical grade biotechnology products. Operated by Leidos Biomedical Research Inc., the BDP facilities include development laboratories, QC analytical laboratories, and GMP facilities, totaling approximately 60,000 sq. ft. The production areas within the GMP facility are designed to ISO 5 (Class 100), ISO 7  and/or ISO 8  specifications to achieve levels appropriate for GMP bulk biologics manufacturing and final filling operations. The BDP includes capabilities in process development, GMP manufacturing, analytical testing, QA oversight, and regulatory strategy  to assure the safety and rights of human subjects for initial clinical trials. They also ensure that the marketing approval of these therapies are based on the demonstration of safety and substantial evidence of efficacy. At least three regulatory issues need to be considered for developing microbial therapies: unintended spreading (shedding) of therapeutic organisms, demonstration of systemic effect of oncolytic viruses, and potential infection following treatment with bacteria-based therapies.Microbial-based therapies often involve live viruses or bacteria that can replicate and thus self-amplify. Thus, one safety concern from a regulatory perspective is that these microbes may be shed from patients and spread to individuals who are in close contact. Although genetic modification may limit the ability of these microbes to be pathogenic, shedding could still occur, particularly for microbial agents that are systemically administered. Therefore, early in the clinical development of microbe-based therapy, it is important that the clinical-trial design includes plans to monitor shedding. Such monitoring not only serves to protect the public health but also provides information for the design of later-phase trials. The details of the monitoring program such as sample collection, monitoring methods, cut-off thresholds and their interpretation, may vary among different microbes. FDA has published a Guidance for Industry to address these issues entitled ‘Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncolytic Products’ . This guOne of the most challenging issues is how to assess the efficacy of intratumorally administered oncolytic viral therapy (IT OVT), a local therapy, in the context of a systemic disease such as metastasis. To address this issue, it is crucial to consider whether such an IT OVT could lead to a systemic treatment effect. The measures to take to demonstrate such an effect could include 1) observation of an abscopal-like effect showing tumor regressions in the lesions that are not injected, in particular, visceral lesions for an OV given to non-visceral lesions; 2) demonstration of prolongation of progression-free or overall survival in patients who have received an IT OVT compared with those who have received control; and 3) demonstration of a systemic immune response. However, the latter could only be used as supportive if it is not accompanied by an improvement in clinical outcome assessment.One of the main concerns of bacteria-based cancer therapy is that these bacteria could cause clinically significant infection and sepsis, especially in cancer patients who may be already immunocompromised due to their underlying disease and/or from concomitant treatment. Some invasive procedures may be needed to administer some of these live bacterial products which may add additional risks to patients. Thus, in early clinical trials design, investigators and sponsors need to consider appropriate plans to mitigate these concerns .We hope that this White Paper will serve to nucleate the growing field of oncolytic microbial therapy. There is much excitement about the potential of this field to treat currently-intractable cancers, and to synergize with other growing modalities such as immunotherapy. In the process of putting this paper together, we saw how many parallels exist between bacterial and viral therapies. We also learned that they both face similar obstacles to commercialization. By harnessing a broad set of mechanisms that cannot be achieved with other strategies, microbial therapies (both viruses and bacteria) have the potential to create the next generation of therapeutics and will accelerate our gains against cancer mortality. Of the many factors that generate enthusiasm about microbial therapies, the ability to specifically target malignant tissue and deliver genetically-engineered payloads are two of the greatest. Achieving these visions will require integration across many disciplines, including oncology, infectious disease, immunology, surgery, manufacturing, preclinical modeling, and regulatory affairs. By providing therapies with unique mechanisms of action, microbial therapies are destined to become integral components of effective cancer management.in situ, without the patient-specific manufacturing complexity, and with the direct tumor-killing ability of an oncolytic virus.T-VEC is a modified herpesvirus with deletion of two viral genes and insertion of GM-CSF. The initial developer of T-VEC was BioVex Ltd, a UK-based company which originated from the academic lab of Robert Coffin at University College London in 1999. It was designed to induce a more systemic benefit than previous oncolytic viruses by providing an immune enhancement component. To increase local anti-cancer effects T-VEC was derived from a higher potency clinical isolate of HSV than had been used before. Two viral genes were deleted: ICP34.5 and ICP47. Deletion of ICP34.5 provides tumor selectivity and deletion of ICP47 leads to increased expression of US11 and usually inhibits antigen presentation. Increased US11 partially overcomes the phenotype of ICP34.5 deletion, which reduces tumor replication, without reducing tumor selectivity. The gene encoding GM-CSF was inserted into the virus to recruit and activate dendritic cells. This was based on the prior observation that tumor-cell-derived cancer vaccines expressing GM-CSF  had demonstrated clinical promise. Previous GVAX vaccines needed to be derived from tumor biopsies (i.e. be autologous), which was logistically challenging. T-VEC was intended to generate an autologous GVAX-like vaccine directly in the patient T-VEC initially began a phase 1 study in the UK in 2003. This study demonstrated that the optimal dosing strategy was a lower initial dose followed by higher subsequent doses. The initial dose served to seroconvert or boost anti-HSV immunity, which then reduced the side effects for subsequent higher doses. These side effects were transient fevers and local reactions. Using this dosing approach T-VEC was well tolerated, and showed promising signs of anti-tumor activity, including necrosis and shrinkage of injected tumors and tumors which were nearby, and inflammation of distant, uninjected tumors . FurtherP=0.051). The improvement in survival was greater in patients without visceral disease  and in previously untreated patients. The trial met its primary objective in the ITT population, and was approved by the FDA for that patient group in 2015. However, the most appropriate patients for treatment with single T-VEC appeared to be those without visceral disease or who were first line. EMA approval was received for these groups in 2016.The Phase 3 study  was condParvoviruses are the smallest viruses currently being developed as oncotherapeutic agents. The rodent protoparvovirus H-1 is currently in Phase I/IIA clinical trials against glioblastoma , 215 andListeria activates the NAPH-oxidase pathway resulting in high levels of reactive oxygen species (ROS) that induce immunogenic tumor cell death , 224. WhClostridium novyi-NT (C. novyi-NT) is an attenuated strain of Clostridium novyi, a spore-forming, Gram-positive, obligate anaerobe that germinates in hypoxic tumor environments. This strain lacks the alpha-toxin gene, which is the major necrotizing toxin responsible for Clostridium species pathogenicity [), C. novyi-NT spores replicate within the hypoxic regions of tumors, eliciting robust tumor lysis in animals including companion dogs bearing spontaneous solid tumors. The anti-tumor effect of C. novyi-NT has also been observed in humans in two clinical trials sponsored by BioMed Valley Discoveries. In a Phase I study of C. novyi-NT in patients with advanced solid tumor malignancies, a single IV injection of C. novyi-NT spores was shown to cause tumor destruction in a metastatic lesion of a patient with colon cancer. In another Phase I study it was determined that a single IT injection of C. novyi-NT is feasible and it led to significant destruction of injected tumor masses in more than a third of patients treated. The most common toxicities are fever and tumor inflammation, which are expected due to the nature of the therapy. Preliminary findings indicate that C. novyi-NT induces a tumor-specific T-cell response. Future clinical studies will build on emerging pre-clinical data of the combination of C. novyi-NT with immune-checkpoint inhibitors.genicity , 77. WheC. novyi-NT germinated exclusively in hypoxic tumor tissues but not in non-malignant hypoxic lesions [C. novyi-NT has been found as well. Mice cured from CT26 tumors by C. novyi-NT were protected from rechallenge with the same tumor cells, indicating underlying immune-based mechanisms [C. novyi-NT kills tumor cells through ROS, immunogenic tumor cell death may have led to epitope spreading and rejection of CT26 tumors.It is worth noting that spores of the obligate anaerobic strain  lesions , 227, unSince its establishment in 1997/1998, the Virotherapy Program at Mayo Clinic has carried out multiple clinical trials using adenovirus, measles virus, reovirus and vesicular stomatitis virus (VSV) platforms. As with many such trials at other Institutions, the road to establishing these trials has typically been very long, expensive and full of new learning opportunities. The overwhelming theme, from the hundreds of patients treated so far, has been that virotherapies have largely proved safe. However, there have been exceptions to this – with one patient treated by intra-tumoral injection of a VSV engineered to express IFN-ß dying days after the virus injection. The cause of this most serious of serious adverse events (SEA) is still being investigated. However, it is clear that virus replication in the liver-metastatic tumor of this patient was very robust and tumor lysis syndrome along with possibly liver-toxic levels of IFN-ß may have contributed. Conversely, although rare, the program has also seen individual cases of great therapeutic success. Thus, one patient with advanced multiple myeloma treated with the highest dose of a recombinant measles virus underwent a dramatic regression of her disease which has proved to be durable out to 4 years following a single-shot treatment. As for the SAE at the other end of the spectrum, it is not completely clear whether this impressive success for virotherapy represents the result of pure oncolysis of the measles virus in the myeloma tumors, immune stimulation following such a high dose of systemically delivered virus or a combination of both.Perhaps the most important lesson that has come out of these hundreds of treated patients and the millions of dollars spent, is that when great successes, or tragedies do happen, it is critical that all such trials are supported by correlative studies put in place to understand the mechanistic basis behind each one. In this light, even a terrible SAE can provide critical information that will help us to advance the field. For example, deep sequencing of a patient’s tumor which supported  abnormally high levels of virus replication can lead to a profile of patient selection criteria which may help to avoid such events in the future. Such information may also help us to develop adjuvant treatments which take tumors predicted to be low-level responders (low virus replication) to a more permissive state. In addition, a full and comprehensive immune-monitoring capability will allow investigators to correlate positive, negative or a lack of clinical responses to immune-virotherapy with host responses, including cytokine profiles. Therefore, it is strongly recommended that comprehensive immune, genomic and virological monitoring assays be put in place before trials recruit their first patients. In this way, the hope is that the current scenario – multiple patients treated with low toxicities and only intermittent responses (good or bad) – may soon be replaced by the preferred situation where the majority of enrolled patients respond well as a result of being pre-selected for specific immune-virotherapies based upon a clear knowledge of their likelihood to respond.Saltikva is attenuated Salmonella armed to produce IL2. It differentiates normal environments from the abnormal, typically hypoxic environments found in solid tumors. There is a preference of varying degrees for different tumor types, but also a clear, ten-thousand-fold preference for tumors over organs such as the healthy liver. Taking residence in tumors and replicating there, this strain of Salmonella incites a host immune response and turns the cancer into a bystander to be destroyed. This ultimately leads to destruction of the tumor, and formation of memory to counter the growth of metastases. Saltikva was not envisaged as a monotherapy. Because it is orally bioavailable and safe, it may synergize with targeted therapies or chemotherapies. Veterinary application of MDB401 has been out-licensed and is on track for market introduction in 2018/19. Next steps in the human development program for Saltikva include clinical trials in the first target, sarcoma of adolescents and young adults.


Almost four years after the discovery of the anabolic action of irisin on bone in mice, ample clinical evidence is emerging in support of its additional physiological relevance in human bone. Irisin inversely correlates with sclerostin levels in adults with prediabetes and with vertebral fragility fractures in post-menopausal women. Furthermore, in athletes we observed a positive correlation between irisin and bone mineral density at different anatomical sites. Our group also described a positive association between serum irisin and bone status in healthy children and multivariate regression analysis showed that irisin is a stronger determinant of bone mineral status than bone alkaline phosphatase. In children with type 1 diabetes mellitus, serum irisin concentrations are positively associated with bone quality and with glycemic control following continuous subcutaneous insulin infusion. Additionally, our in vitro studies suggest the existence of a negative interplay between PTH and irisin biology and these results were also supported by the observation that post-menopausal women with primary hyperparathyroidism have lower levels of irisin compared to matched controls. In this review, we will focus on recent findings about circulating level of irisin in different populations of human subjects and its correlation with their bone status. Skeletal muscle activation is a key aspect that improves quality of life in individuals who engage in physical activity because of stimulation of energy metabolism and synchronous reinforcement of bone and muscle mass. Large prospective studies have shown that running and walking activities reduce mortality by 20–40% ,2 and slLately, a growing body of evidence suggests that muscle talks to bone not just through mechanical signals, but also via a finely tuned network of molecules termed myokines . They arThe myokine irisin is cleaved from its precursor fibronectin type III domain-containing protein 5 (FNDC5) and secreted into the bloodstream. Irisin has been shown to stimulate white adipose tissue (WAT) to adopt a brown adipose tissue-like phenotype through increasing cellular mitochondrial density and expression of uncoupling protein-1. This trans-differentiation process triggers thermogenesis, increasing energy expenditure and improving glucose homeostasis . HoweverThe skeletal effects of intermittently administered irisin have been studied in healthy mice and osteoporotic murine models. Treatment of healthy mice with recombinant irisin improves bone mass and its geometric and biomechanical properties compared to mice treated with vehicle . FurtherIn humans, irisin correlates inversely with serum sclerostin levels  and in pIn this review, we will focus on these recent findings about circulating levels of irisin in different populations of human subjects and its correlation with their bone status.Although it has been clearly established that irisin serum concentration increases in response to exercise, the influence of exercise intensity (high-intensity or continuous-moderate) on irisin secretion remained uncertain. Some data suggest a greater increase following acute exercise compared with aerobic exercise , with a Aiming to understand a possible influence of rising levels of irisin on increased bone mass in athletes, we investigated the correlation between its serum levels and total and sub-regional BMD in Caucasian football players of the Italian championship. Our results showed a positive correlation between irisin and total body BMD. Furthermore, linear association was also detected at specific bone-sites such as the right arm, lumbar vertebrae and head . We did Beside the type of sport activity, also a day-night fluctuation may affect irisin synthesis in humans. Anastasilakis et al. performed an observational, cross-sectional study on 122 healthy, young individuals divided in subgroups subjected to day-night rhythm, standardized meal ingestion and 30-min aerobic exercise. Authors reported a day-night rhythm of irisin secretion, with its lowest levels early in the morning and its peak levels around 9:00 pm. Moreover, although authors showed that irisin was not affected by previous physical activity habits, they found higher circulating levels after short-term aerobic exercise .Early childhood and adolescence are periods of rapid bone growth and the achievement of peak bone mass is a crucial determinant of lifelong skeletal health ,26,27. EInterestingly, we found that irisin was positively correlated with circulating osteocalcin, the osteoblast-derived protein, which has been recently defined the bone-hormone that enhances energy metabolism, skeletal muscle function and brain development . We alsoMetabolic syndrome (MetS) is a clinical condition in which a set of risk factors, including obesity, hypertension, dyslipidemia, hyperglycemia and insulin resistance, predispose to cardiovascular diseases, diabetes and osteoporosis ,40. SeveOur study was the first 6-month randomized trial monitoring irisin serum levels and the adherence to the diet throughout the trial. However, two previous studies on irisin and MetS, which were based on shorter dietary interventions, showed opposite results. A randomized, controlled trial, which compared the effects of two different 2-month-long hypocaloric dietary interventions, the control and RESMENA diets , reportePatients affected by primary hyperparathyroidism (PHPT), an endocrine disorder characterized by excessive release of parathyroid hormone (PTH), experience reduction of BMD, particularly at the cortical site of the distal radius . Biomolein vivo. Authors also provided evidence that irisin treatment upregulates the expression of sclerostin in osteocyte-like cells (MLO-Y4) and increases serum sclerostin levels in a dose dependent manner when injected in continuous manner once a day for 6 days in mice [Very recently Kim et al. identified integrin αV/β5 as the receptor for irisin on osteocytes . The ide in mice . These r in mice  and with in mice . Further in mice . These u in mice . Therefo in mice ,11 (FiguAlmost four years after the discovery of the anabolic action of irisin on bone in mice , several