Osteosarcoma is a malignant primary tumor of bone, arising from transformed progenitor cells with osteoblastic differentiation and osteoid production. While categorized as a rare tumor, most patients diagnosed with osteosarcoma are adolescents in their second decade of life and underscores the potential for life changing consequences in this vulnerable population. In the setting of localized disease, conventional treatment for osteosarcoma affords a cure rate approaching 70%; however, survival for patients suffering from metastatic disease remain disappointing with only 20% of individuals being alive past 5 years post-diagnosis. In patients with incurable disease, pulmonary metastases remain the leading cause for osteosarcoma-associated mortality; yet identifying new strategies for combating metastatic progression remains at a scientific and clinical impasse, with no significant advancements for the past four decades. While there is resonating clinical urgency for newer and more effective treatment options for managing osteosarcoma metastases, the discovery of druggable targets and development of innovative therapies for inhibiting metastatic progression will require a deeper and more detailed understanding of osteosarcoma metastasis biology. Toward the goal of illuminating the processes involved in cancer metastasis, a convergent science approach inclusive of diverse disciplines spanning the biology and physical science domains can offer novel and synergistic perspectives, inventive, and sophisticated model systems, and disruptive experimental approaches that can accelerate the discovery and characterization of key processes operative during metastatic progression. Through the lens of trans-disciplinary research, the field of comparative oncology is uniquely positioned to advance new discoveries in metastasis biology toward impactful clinical translation through the inclusion of pet dogs diagnosed with metastatic osteosarcoma. Given the spontaneous course of osteosarcoma development in the context of real-time tumor microenvironmental cues and immune mechanisms, pet dogs are distinctively valuable in translational modeling given their faithful recapitulation of metastatic disease progression as occurs in humans. Pet dogs can be leveraged for the exploration of novel therapies that exploit tumor cell vulnerabilities, perturb local microenvironmental cues, and amplify immunologic recognition. In this capacity, pet dogs can serve as valuable corroborative models for realizing the science and best clinical practices necessary for understanding and combating osteosarcoma metastases. Table 2), and justify the exploration of precision medicine approaches for improving survival outcomes. To accelerate discovery to impact, the rational development of anti-metastatic therapeutics requires a convergent science approach including (1) a better understanding of OS metastasis biology in relation to the lung microenvironment and (2) the availability of engineered and natural model systems that most faithfully recapitulate the complexities of metastatic progression. Through transdisciplinary collaborative research, it is envisioned that novel and effective anti-metastatic therapeutics can be identified and translated to extend the lives of patients with OS by eradicating or thwarting the progression of subclinical micrometastatic disease that persisted following standard multi-agent chemotherapy.Since the institution of chemotherapy in the 1960s, relapse-free survival for osteosarcoma (OS) patients with localized disease has dramatically improved. The current standard of care involves surgical resection of the primary tumor and multi-agent chemotherapy (both in the neoadjuvant and adjuvant setting) which can result in 5-year survival rates up to 70% for patients with localized disease . The negmetastatic cascade, describes the progressive steps of tumor cell dissemination from the primary tumor, transit within the blood vasculature, and the establishment of clinically detectable pulmonary metastases    and seve c-FLIP)  and OS c c-FLIP) , 48, 49.2) or Dynes per centimeter squared (Dyn/cm2)  . FSS in location . Lien an chamber . The aut chamber , exhibitIf OS cells can resist anoikis and adapt to damaging FSS in the blood circulation, arrest, and survival in the lung microvasculature presents the next significant challenge to metastatic OS cells. Several studies using the experimental metastasis model (tail vein injection of tumor cells) have demonstrated that the majority of tumor cells that arrive in the lung do not survive, and only a small subset of the initial population (1–6%) were able to successfully establish metastases , 32. TheRedox stress is a major microenvironmental stressor that contributes to tumor cell clearance in the lung since several studies have provided microscopic imaging evidence and “omic” data supporting this notion. Qiu et al.  have sho2+ concentration within ER lumen, and ATP supply (Another type of cellular stress closely linked to redox stress is endoplasmic reticulum (ER) stress. Protein folding processes within the ER are exquisitely sensitive to perturbations in cellular redox state, CaP supply . Redox sP supply . The unfP supply . The UPRP supply , 70. SevP supply , and shRP supply , were foP supply . DownstrP supply . SeveralP supply –75.in vitro. Additionally, Macklin et al.  interactions between OS cells, ECM, and lung parenchymal cells, model systems that can maintain or partially recapitulate some aspects of these 3D interactions will allow researchers to interpret metastatic OS cell responses to gene therapy or pharmacologic intervention in a relevant microenvironmental context. Indeed, several studies have demonstrated that tumor cell response to therapeutics differ when comparing 2D vs. 3D growth conditions –86. To tex ovo), where xenograft human tumor fragments or a tumor cell suspension  relatively inexpensive, and (3) can be used for high-throughput screening of targeted therapies. Disadvantages of the CAM model include: (1) short observation times (days), (2) inability to study tumor cell interactions with the immune system since the chick CAM is immunodeficient until developmental day 18  is a highly vascularized membrane that primarily functions as a gas-exchange organ for the developing embryo . The CAMspension  can be pspension , 91, tumspension , intravaspension , tumor cspension  94–98),–98,ex ovspension . In OS rspension , angiogespension , and metspension . The mail day 18 , and (3)l day 18 . The chil day 18 , interacl day 18  since thex vivo, lung tissue explant model where fluorescently labeled tumor cells in viable lung tissue , first developed by Mendoza et al. , and furg tissue  can be mlsewhere , retain vo lungs . The PuMvo lungs , 25, 106vo lungs . For imavo lungs . One majvo lungs . If a reex vivo perfusion model where the lungs were removed en bloc and imaged on an inverted microscope. Intravital imaging of the microcirculation of various organs (such as lung or liver) was reported by Varghese et al.  those that spontaneously develop primary tumors and subsequently develop metastasis, (2) those that are implanted orthotopically  with spontaneous distant metastases , and (3)Traditionally, the oncogenic transformation and malignant behaviors of cancer cells have been ascribed to perturbations involving multiple and interactive molecular factors rooted in genetic alterations and dysregulated biochemical signaling. While many aspects of cancer cell phenotype, including metastasis, can be adequately characterized and studied through the singular lens of biology, there is overwhelming evidence that mechanical forces exerted by and upon cancer cells, surrounding stromal elements, and ECM are integrally linked with oncologic activities, including cancer cell invasion and metastasis , 125. Liin vitro biomimetic platforms currently used in cancer research are summarized, and their suitability for studying unique aspects related to OS metastasis are highlighted.While a preponderance of cancer investigations includes studies based on two-dimensional (2D) cell models, such experimental methods that rely upon cancer cells grown in monolayer do not recapitulate the true interactions between cells-cells and cells-extracellular matrices encountered during solid tumor formation, evolution, and metastatic progression. The bidirectional interactions of cancer cells with the tumor microenvironment generates biological complexity, which can be more thoroughly studied through three-dimensional (3D) modeling strategies that include biomimetic engineered tumor models. Through the purposeful design of various mechanical platforms, it is now possible to ask and answer specific questions regarding how cancer cells respond to highly tunable variables including matrix stiffness, interfacial geometry, cell curvature, and other mechanotransduction gradients –131. By Tumor cell masses naturally grow in 3D and cellular behaviors are dependent upon multiple biochemical and mechanical cues heterogeneously distributed throughout the microenvironment . Compared to conventional 2D cell culture methods, spherical 3D tumor models are superior for recapitulating the spatial cellular and biochemical heterogeneity of solid tumors. Tumor spheroids are cancer cell aggregates ranging in size from 20 to 1,000 μm in diameter and can be formed through various techniques, with the easiest method reliant upon cell buoyancy . AdditioSpecific for OS, 3D culture systems with spheroids have been utilized for the past 2 decades for studying the effects of the tumor microenvironment on various aspects of OS biology and has been thoroughly summarized by De Luca et al. . DerivedThe ECM is critical in shaping tumor biologic responses through mechanotransducive mechanisms, and the investigation of cancer cells embedded within scaffold-based constructs that vary in chemical composition, shape, density, structure, and porosity allows for researchers to dissect differential mechanotransductive contributions for the induction of diverse malignant phenotypes and cellular processes displayed by cancer cells. Scaffolds can be constructed from either natural or synthetic polymers, with both sharing conserved properties of biocompatibility and promotion of cellular adhesion. Natural scaffold materials are typical ECM proteins while synthetic scaffolds are derived from tunable and crosslinking materials including polyethylene glycol (PEG) and polylactide-co-glycolide acid (PLGA), as well as porous ceramic biomaterials such as bioactive hydroxyapatite and tricalcium phosphate.in vivo microenvironment of nascent tumor mass growth in 3D . Structurally, microfluidic systems can be fabricated to include diverse shapes on a micro- or nanoscale including channels and chambers with highly precise diameters, shapes, and flow control rates. When combined with 3D cell culture systems such as spheroids, microfluidic platforms can recapitulate diverse complex processes representing different stages of the cancer progression including tumor-vascular interface responses, diffusional effects of biomolecules on cell populations, and pathologic cancer processes including invasion, angiogenesis, and metastasis –170. RecConventional OS models for studying experimental therapies most frequently are reliant upon xenogeneic and syngeneic transplant models conducted in mice, however, the inclusion of complementary model systems  have gained wider appeal and scientific acceptance for improving predictive modeling of cancer biology and metastasis. While xenogeneic models, including patient derived xenografts, may provide information pertaining to the sensitivity of human OS tissues or cell lines to specific therapeutics, tumor-host interactions  are poorly recapitulated in comparison to what occurs in people who develop OS spontaneously. Although syngeneic models more accurately represent immunologic tumor-host responses than xenogeneic systems, the process of tumor formation and spontaneous metastasis in any transplant model remains artificial, likely underestimating the complexity for how OS naturally progresses in an immunocompetent host. To accurately identify and expedite the clinical translation of novel therapeutics to people with metastatic OS, the evaluation of experimental strategies, in particular immune-based, should be conducted in the most highly relevant and immunocompetent tumor model.Besides people, canines are the only other large mammal that spontaneously develops OS with substantive frequency. Canine appendicular OS is the most common primary bone tumor in dogs of large to giant skeletal size, and has been estimated to affect at least 10,000 pet dogs every year in North America , which iThe current standard of care for human patients diagnosed with OS remains largely unchanged from that first used in the early 1980s , being nThe factor that most strongly influences outcomes in human patients is the presence or absence of metastatic lesions, usually of the lung parenchyma. Patients who develop lung metastases, whether at diagnosis or years after completing therapy, face a dismal prognosis, with fewer than one in five patients surviving more than 5 years beyond this event , 191. MuPatients with both resectable and unresectable metastatic disease at relapse are usually offered systemic therapy, most commonly with high dose ifosfamide  or multiAs mentioned previously, each step of the metastatic cascade is a rate limiting step. For example, if a new drug can prevent OS cells from leaving the primary tumor, invading local tissue, or entering local blood vessels, then the metastatic cascade is stopped in its tracks. Indeed, every step of the metastatic cascade harbors several druggable targets in various types of cancer, as summarized in Successful dissemination and colonization of distant tissues by a tumor cell requires navigating a gauntlet of interactions with normal cells and associated tissues . Each inStromal elements produced by both host cells and tumor cells may play a particular role in the survival of metastasis-initiating cells and in the maintenance of their stem-like features. Zhang and colleagues recently showed that FGF signaling within the metastatic environment triggers a fibrogenic program within disseminated tumor cells that promotes their stemness and survival . SignalsThe laboratories of Roberts and Cam have identified targetable bi-directional signaling between OS cells and lung epithelial cells that appears critical for metastatic colonization. Using a combination of human tissues, xenografts, syngeneic mouse models, and canine models of disease, they have shown how a ΔNp63/IL6/CXCL8 signaling axis mediates tumor-host signaling events critical to the metastatic process. In their model, tumor cells primed by aberrant expression of ΔNp63  an alte. DisruptSignals that facilitate tumor cell survival within the metastatic niche can emerge from either lung-resident cells or from cells that invade that niche, often in response to tumor-derived signals. For example, Baglio and colleagues have shown that TGFβ expressed on the surface of extracellular vesicles from OS cells can also elicit production of large amounts of IL6 by mesenchymal stem cells . The relSome tumor-host interactions prove detrimental to the survival of disseminated tumor cells. Kleinerman's group has made a series of observations that suggest most disseminated tumor cells that reach the lung will be eliminated through activation of a suicide signal when the FAS receptor expressed on the surface of the tumor cells engages FAS ligand, which is expressed constitutively within the lung . This phSearching for epigenetic changes that facilitate metastatic colonization of lung tissue by OS cells, Morrow and colleagues recently identified genetic loci that acquire enhancer activity in cells with high metastatic potential . Among gThe unique metabolic demands of the primary tumor vs. metastasis are reflective of their different microenvironments (cellular and extracellular components), nutritional availabilities, and level of oxygenation. A rapidly growing primary tumor mass requires a constant supply of energy (ATP) and biomacromolecules  . During 2−), can modify mitochondrial DNA, which in turn, can negatively affect the electron transport chain (ETC), mitochondrial membrane potential, and ATP production (− can irreversibly inhibit complex I of the ETC (−), another potent RNS, can inhibit multiple enzymes in the mitochondria such as complexes I–IV, as well as aconitase of the tricarboxylic acid cycle  and reactive nitrogen species (RNS) produced by the lung parenchyma can affect tumor cell mitochondrial function in a number of ways , 212. Fooduction . Prolong the ETC . Peroxynid cycle . Metabolid cycle , melanomid cycle , and ostid cycle . For exaid cycle . Shuttliid cycle , 217. Otid cycle . These oid cycle , as wellid cycle . Collectid cycle . FurtherRecently, immunotherapy has been heralded as a breakthrough for the management of diverse liquid and solid tumors, and its ascension as a major therapeutic pillar is underscored by a rapidly increasing number of FDA approved immune-based treatments for cancers that are resistant to conventional modalities. The anticancer activities of immunotherapies can be ascribed to the cooperative effector functions exerted by both the innate and adaptive immune arms, and while immunotherapy is highly effective for certain solid tumors like melanoma, renal cell carcinoma, and others, its promise for benefiting patients diagnosed with metastatic OS remains largely disappointing to date –223. Par+; pro-inflammatory) rather than a M2-subtype profile  are associated with better overall survival in OS patients. Incongruent findings among studies regarding the role of TAMs in OS biology could be related to the inherent limitations of single timepoint tissue assessments which fail to capture the dynamic nature of immune cell infiltration within the tumor microenvironment. Nonetheless, the majority of histologic findings provide supportive justification to therapeutically manipulate TAMs profiles within OS lesions that have potential to either favor immune activation (Mifamurtide)  and T lymphocytes, respectively. Within primary OS lesions, tumor-associated macrophages (TAMs) that can be distinguished via genomic signatures, cell surface markers, and functional activities (inflammation vs. immunosuppression) have received considerable attention for their prognostic value and functional role in OS metastasis –230. Mosmurtide)  or inhibmurtide) , 233, fo+) phenotypes participate in shaping immunosurveillance of OS lesions  and their contribution to metastasis immunobiology. Analyses of TILs within the OS microenvironment have shown that both effector and suppressor T lymphocyte .Streptococcus pyogenes and Serratia marcescens injections, termed Coley's toxin (Immunomodulatory agents modify immune responses by amplifying the recognition of cancer cells (immunostimulation) or by attenuating the immunosuppressive activities exerted by cancer cells within the local tumor microenvironment. The innate arm of the immune system comprised of natural killer cells, macrophages, dendritic cells, and primordial T cell subsets  are predominant effector targets of immunomodulatory strategies. The clinical significance of immunomodulatory interventions relevant to sarcomas was noted over a century ago, when William Coley in 1891 reported objective responses in a small minority (10%) of patients with non-resectable sarcomas (bone and soft tissue) treated with heat-inactivated 's toxin . The pot's toxin , 240, an's toxin .in vitro and in preclinical investigations for activating monocytes and macrophage to a tumoricidal state  and cytokine-based therapies. Based upon its mechanism al state , 243, asal state , clinicaal state . Additioal state , furtheral state . While eIn the setting of macroscopic OS metastases, the tolerability and potential benefit exerted by exogenous IL-2 has been explored. In one study, Meazza and colleagues reported the outcomes of 35 pediatric OS patients with macroscopic OS treated with surgery and combinatorial chemoimmunotherapy comprised of IL-2, MAP, ifosfamide, and lymphokine-activated killer (LAK) cell infusion. While the study was not designed to determine the immunobiologic benefit derived from IL-2 and LAK cell infusion, adverse effects associated with IL-2 therapy were tolerable (grade I and II) with most common side effects being fever, flu-like symptoms, hypotension, and cytokine release syndrome . In a diThe engineering of monoclonal antibodies to enhance the immune system's attack on cancer cells has dramatically expanded therapeutic options for multiple hematopoietic and solid tumor histologies, and currently over a dozen antibodies have received FDA approval for treating different cancers . Upon biIn addition to monoclonal antibodies that target OS cells, considerable focus has been on manipulating tumor-specific or tumor microenvironmental (TME) cues with antibody strategies, specifically enhancing the quantity and quality of intratumoral infiltration with immune cell populations through blockade of checkpoint signaling . CheckpoTumor vaccines are an active form of immunotherapy in which robust adaptive immunity is developed and cytotoxic T lymphocytes are generated for recognizing and killing cancer cells. The induction of antitumor responses against known or unidentified tumor antigens can be achieved through vaccine strategies inclusive of whole cells, lysates, proteins, DNA, RNA, or peptides. For vaccine strategies to be effective, dendritic cells must present tumor peptides within major histocompatibility complexes, as well as costimulatory signals, to fully activate the effector functions of cytotoxic T lymphocytes. Given the key role of dendritic cells for initiating the activation and proliferation of cytotoxic T lymphocytes with anticancer effector functions, dendritic cell vaccines have been explored in patients with metastatic OS through the conductance of two recent clinical studies , 266. MiListeria monocytogenes which expresses three immunodominant epitopes of HER2 has been evaluated for in patients with HER2-expressing solid tumors (NCT02386501). This vaccine construct (ADXS31-164) was granted Fast Track designation by the FDA for treatment of patients with newly diagnosed, non-metastatic, surgically-resectable OS, and patients will be treated with this vaccine strategy through the Children's Oncology Group consortium. As a predecessor to the pediatric trial, ADXS31-164 was piloted in pet dogs with OS and provided valuable translational information. Mason and colleagues demonstrated that treatment with ADXS31-164 induced HER2-specific immunity in 15/18 dogs and resulted in a significant increase in median disease-free interval (615 days) and median survival (956 days) when compared to a historical control group. Overall survival rates at 1, 2, and 3 years for dogs treated with ADXS31-164 were 78, 61, and 50%, respectively  or adaptive (cytotoxic T lymphocytes) immune effector cell populations that have been genetically manipulated. The infusion of cellular therapies, specifically cytotoxic T lymphocytes into tumor-bearing patients, circumvents the reliance of vaccine strategies to successfully active a large population of tumor-specific effector lymphocytes in the recipient host. For T-cell based strategies, adoptive cell therapies rely upon the genetic engineering of T lymphocytes to either express a known T cell receptor (transgenic TCR) or chimeric antigen receptor (CAR), with both strategies resulting in the production of T cells with defined specificity, which can be MHC-restricted (transgenic TCR) or -unrestricted (CAR). While CAR technology has clinically impacted the management of hematopoietic cancers such as acute lymphoblastic leukemia and diffuse large B-cell lymphoma, the application of CARs for effectively treating OS metastases remains incompletely defined. Recently, the results of a phase I/II clinical study evaluating the tolerability and activity of HER2-CAR T cells were reported by Ahmed and colleagues. In this study, 16 patients with recurrent and/or metastatic OS were treated with escalating intravenous doses of T cells expressing an HER2-specific CAR with CD28ζ signaling domain . Treatmeex vivo, and then delivered back to dogs by intratumoral injection following the completion of radiation therapy. Of the 10 dogs treated, 5 remained metastasis-free at 6 months, and one had regression of a suspicious pulmonary nodule detected at the time of diagnosis. While preliminary in nature, these studies in canine OS provide the underpinnings to prospectively evaluate different combinatorial strategies inclusive of adoptive cell therapies for combating OS metastatic progression.The development and clinical assessment of adoptive cell therapies have been piloted in canine OS, providing valuable preclinical data regarding feasibility and activity. For CAR T cells, HER2 has also been explored as a target for canine OS, and Mata and colleagues reported the successful development of HER2-CAR T cells that killed HER2+ canine OS cell lines in an antigen dependent manner . AdditioIn order for animal models of human pathologies to be useful and informative, it is necessary for the experimental system to be readily available and accurately recapitulate the natural course of disease. For OS, where the genetic underpinnings are chaotic and the etiopathogenesis remains elusive, the creation of experimental model systems becomes fundamentally difficult, and potentially flawed; as the blueprint of models are derived from and constructed upon current understandings of disease processes. Pet dogs that spontaneously develop OS have potential to serve as excellent naturally-occurring models for diverse comparative pathologies, including OS. Through collaborative research efforts, canine OS can be uniquely positioned in the scientific discovery pathway for understanding metastasis biology, which can drive and accelerate the identification of new promising anti-metastatic therapies. Several characteristics of canine OS are noteworthy and with the continued inclusion and future innovative addition of pet dogs as comparative OS models, it would be expected that significant progress will be made toward improving the outcomes of patients diagnosed with OS metastases.Pediatric OS, by definition is categorized as a rare tumor . For localized OS, surgery and MAP chemotherapy in pediatric patients produces 5-year event free survival in the majority (60–70%) of patients treated. In this good responder population with favorable histologic Huvos grade, the opportunity to clinically evaluate and realize the anti-metastatic activity of novel therapies is narrow and temporally protracted. While patients with recurrent and/or metastatic OS can be readily included for investigating novel therapies, biologic responses to experimental agents might differ between macro- and microscopic disease settings, and confound the accurate identification of new agents with reproducible anti-metastatic properties.Canine OS is the most common primary bone tumor in large and giant breed dogs, and has been estimated to affect at least 10,000 pet dogs every year in North America , which i3 difference in anatomic size between mice (20 g) and humans (70 kg). Specifically for the evaluation of novel drugs or drug delivery strategies for combating OS metastatic progression, differences in anatomic size and corresponding dimensions of metastatic lesions can strongly bias treatment outcomes simply as a function of tumor volume and interstitial pressures, as therapeutics released into the pulmonary parenchyma are still governed by the physical laws of diffusion prior to reaching their intended targets, being OS metastatic foci. Fick's law of diffusion states that the rate of movement (mass flux) can be modeled mathematically by the following equation:Despite their accepted research value, some limitations of murine models remain irreconcilable including the >10J represents mass flux, D represents molecular diffusivity of a specific therapeutic agent within a microenvironment, ΔC represents the change in concentration gradients, and Δx represents distance of diffusion. Although several determinants influence mass flux J (representing the movement of therapeutic agents), diffusional distances within a chosen experimental system directly affects the movement rates of therapeutics, thereby having the potential to either positively  or negatively (large anatomic size and expansive diffusion distances) bias treatment outcomes. As such, drugs that demonstrate potent activity in murine models of OS metastasis can be partially attributed to the diminutive diffusion distances required to be traversed by therapeutic agents. However, when translated to larger mammals including human or canine OS patients, where diffusion distances are log orders greater, achieving therapeutic response becomes more difficult  and prostate , basic metastasis research in OS (~860 entries) is taking its first furtive steps. Discovering new and critical processes that contribute to OS metastasis biology will pave the way for the development of novel anti-metastatic therapies that can be integrated in the current standard of care. Paradigm shifts in animal research should include a focus on lung colonization, clearing or halting the progression of micrometastases, and shrinkage of established metastases as being translational targets, as recognized and supported by leaders in the OS field , 281. FuFueled by increasing commitments of resources, veterinary oncology collaborative groups have rapidly become more organized, more agile, and more capable of efficiently conducting high-value pilot studies, complex biology studies, and large clinical trials in client-owned pets that develop cancer. The Comparative Oncology Trials Consortium, headquartered at NIH, has undertaken several initiatives to broaden the comparative biology of human and canine cancers and to improve the efficiency with which they can generate pharmacokinetic, pharmacodynamic, and initial efficacy data in canine clinical investigations that can guide human trial development. Recent initiatives arising from the NIH Cancer Moonshot program have recognized opportunities for incorporating canine clinical trials into the evaluation of immune-oncology approaches by funding several large grants and spawning new comparative immuno-oncology consortia.Intelligent use of the data arising from this work should improve the likelihood of good outcomes in human clinical trials. Coordination across canine clinical consortia, basic scientists, and industry has become increasingly important. The ability to share data and to plan collaboratively has been enhanced by integration of veterinary oncologists into pediatric clinical trials groups and vice-versa. Efforts led by a handful of philanthropic groups to intensify dialog between these stakeholders have met with increasing interest and engagement. These achievements to date represent only initial forays into truly integrated drug development and science—with opportunity far surpassing the current actuality.With continued and growing interest of the scientific community for including spontaneous tumor models to accelerate novel drug development, comparative oncology centers of excellence must keep pace and commensurately grow to meet the expectations of an ever increasing desire for executing high-value clinical trials in an agile and efficient manner. To achieve these deliverables, existing comparative oncology centers must expand capacity, and new centers must develop. Incentivization for such development can be achieved through different mechanisms including active participation in Clinical and Translational Science Awards (CTSA) program and integration with existing Basic or Comprehensive Cancer Centers.Central coordination and unification across participating comparative oncology centers should be strongly advocated, thereby increasing the efficiency and impact of ongoing and future prospective clinical trials. Regional consortia operating in silos will not likely maximize collaborative efforts, and have potential to dilute limited shared resources and manpower. Visionary and inclusive leadership in this arena is necessary, and will ensure that all participating teams can be maximally and directionally aligned for the greatest translational impact as possible.Cancer metastasis remains the leading cause of mortality for people afflicted with diverse solid tumor histologies. While tremendous advances in therapy have been achieved over the past decade for some tumor types, the management and outcomes of aggressive sarcoma metastases, including OS, remains almost at a standstill. To impact the lives of patients suffering from OS metastases, it will be necessary to deepen our fundamental understanding of OS metastasis and its specific vulnerabilities at both the cellular and microenvironmental levels. Additionally, the translation of new and promising therapeutic discoveries must be evaluated using complementary model systems that faithfully recapitulate natural metastatic disease progression in people. Given the conserved biology of OS in humans and dogs, unique opportunities exist for human and comparative oncology researchers to engage in translationally impactful collaborations, which uniquely include pet dogs with OS to expand the understanding of metastasis biology and clinically realize the activity of novel investigational therapeutics that target OS metastatic progression.All animal experiments were done with ethics approval of the local Animal Care Committees.TF, RR, and ML: project conception and manuscript authorship.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.


Understanding pharmacokinetics and biodistribution of antibody–drug conjugates (ADCs) is a one of the critical steps enabling their successful development and optimization. Their complex structure combining large and small molecule characteristics brought out multiple bioanalytical methods to decipher the behavior and fate of both components in vivo. In this respect, these methods must provide insights into different key elements including half-life and blood stability of the construct, premature release of the drug, whole-body biodistribution, and amount of the drug accumulated within the targeted pathological tissues, all of them being directly related to efficacy and safety of the ADC. In this review, we will focus on the main strategies enabling to quantify and characterize ADCs in biological matrices and discuss their associated technical challenges and current limitations. Staphylococcus aureus bacterial infection . Fo. Fo87]. al model  or to asomponent ,91,92,93omponent   [Surprisingly, though the dual radiolabeling of both ADCs components should provide the most comprehensive understanding of ADCs in vivo performances, only a very few examples have been reported to date ,104,105.led MMAF , and a 8analogue . DigitalAlthough allowing a high sensitivity of detection, quantitative radio imaging studies provide no information on the identity of ADC-related species and drug metabolites present in the tissues. To this purpose, these imaging methods must be associated to qualitative bioanalytical methods. In this respect, tissues extraction followed by a SEC-HPLC or standard HPLC analyses coupled to a radioactivity detector can partly circumvent these issues ,101. FurWhether it refers to single or dual labeling, these methods all require expensive radioisotopes and dedicated facilities that complicate their clinical transfer. In addition, the radioactive tags are often incorporated randomly within the ADCs core, with potential consequences on their in vivo behavior. In this respect, the incorporated tag may cause immunoreactivity or alter the characteristics of ADCs binding and distribution.ADME characterization of ADCs is a complex process due to the multicomponent and heterogeneous nature of these conjugates. While conventional large- and small-molecule methods can be used for ADC bioanalysis, it is however important to understand their limitations to develop ADCs-dedicated bioanalytical strategies.These recent years, the hybrid LB-LC–MS method has become the most versatile assay in ADCs bioanalysis since they can provide information not only on the entire molecule but also on ADC-derived analytes. When combined with a highly specific LC–MS method, LBA has overcome its intrinsic limitations, mainly related to interferences with highly abundant proteins, to enable unambiguous identification of ADC-derived analytes in many different biological samples. In addition, recent emerging technologies such as an enrichment strategy via pH regulation and ionic solvent strengths  are compThe use of radiolabeled ADCs to support pharmacokinetic and biodistribution studies is undoubtedly a way to reach high detection threshold. More specifically, designing a dual radiolabeled ADC, harboring distinct radioactive tags on the protein component and the small molecule drug, could appear as a particularly valuable strategy to monitor the in vivo fate of both entities simultaneously and to accurately and sensitively measure their relative amount during the circulation and distribution phases. Combined with whole-body imaging, such an approach can provide useful information on overall stability of the conjugate as well as its biodistribution within target-expressing and nontarget-expressing tissues, thus assessing the ADCs efficacy and potential toxicity. However, imaging and accurate quantification by radioactive counting must be associated to qualitative analyses such as HPLC or SEC-HPLC to provide information about the identity of the radio emitting species. Furthermore, the dual radiolabeling must neither interfere with the physicochemical properties of both ADCs components, nor add further heterogeneity that could alter ADCs binding properties and in vivo behavior. Accordingly, the conception of dual labeled ADCs must imperatively rely on efficient radiolabeling reactions that do not affect the drug properties, namely traceless reactions, and on site-specific conjugation reactions aiming at controlling the modification made within the antibody core. Once these limitations are overcome, the dual labeling strategy could become a central methodology to rapidly challenge and optimize various ADCs in preclinical models, thus accelerating their transfer to clinical studies.


Mild traumatic brain injury (mTBI), or concussion, occurs following a direct or indirect force to the head that causes a change in brain function. Many neurological signs and symptoms of mTBI can be subtle and transient, and some can persist beyond the usual recovery timeframe, such as balance, cognitive or sensory disturbance that may pre-dispose to further injury in the future. There is currently no accepted definition or diagnostic criteria for mTBI and therefore no single assessment has been developed or accepted as being able to identify those with an mTBI. Eye-movement assessment may be useful, as specific eye-movements and their metrics can be attributed to specific brain regions or functions, and eye-movement involves a multitude of brain regions. Recently, research has focused on quantitative eye-movement assessments using eye-tracking technology for diagnosis and monitoring symptoms of an mTBI. However, the approaches taken to objectively measure eye-movements varies with respect to instrumentation, protocols and recognition of factors that may influence results, such as cognitive function or basic visual function. This review aimed to examine previous work that has measured eye-movements within those with mTBI to inform the development of robust or standardized testing protocols. Medline/PubMed, CINAHL, PsychInfo and Scopus databases were searched. Twenty-two articles met inclusion/exclusion criteria and were reviewed, which examined saccades, smooth pursuits, fixations and nystagmus in mTBI compared to controls. Current methodologies for data collection, analysis and interpretation from eye-tracking technology in individuals following an mTBI are discussed. In brief, a wide range of eye-movement instruments and outcome measures were reported, but validity and reliability of devices and metrics were insufficiently reported across studies. Interpretation of outcomes was complicated by poor study reporting of demographics, mTBI-related features , and few studies considered the influence that cognitive or visual functions may have on eye-movements. The reviewed evidence suggests that eye-movements are impaired in mTBI, but future research is required to accurately and robustly establish findings. Standardization and reporting of eye-movement instruments, data collection procedures, processing algorithms and analysis methods are required. Recommendations also include comprehensive reporting of demographics, mTBI-related features, and confounding variables. Eye movements are the basis of how humans gather information about the environment, which is then used to allow the perception of vital information needed for safe navigation or task performance. Eye movements have been investigated via various methods since the 1700s   eye-tracking instrumentation used to examine people following an mTBI compared to healthy controls; (2) commonly reported eye movement outcomes from eye-tracking studies; (3) mTBI specific influences on these eye movement outcomes; and (4) recommendations concerning future protocols.The key terms were “mild Traumatic Brain Injury,” “Eye movement,” and “Eye-tracking.” A list of synonyms was created for each key term . Key terAn initial title screen for relevant articles was performed by the reviewer (SS) once the searched database results had been combined. After the initial title screen, both the titles and abstracts of the selected articles were reviewed by three independent reviewers . A review of the full text was required if it was not clear from the title or abstract whether the study met the review criteria.Articles were included if they reported use of an eye-tracking measurement instrument to quantify eye movements  in people with mTBI. Studies were included only if they tested a healthy control cohort or used a baseline pre-injury test as a control for comparison with mTBI cohorts so that injury-specific differences could be identified. If articles including another clinical cohort  and were synthesized into table format by the reviewer (SS) and a second reviewer (LP) confirmed the entered data –3. Data The search strategy yielded 86 articles, excluding duplicates  investigated healthy controls and mTBI with average age ranges between 13 and 39 years old .There was also very poor reporting of specific data processing or analysis methods used to derive the outcomes of interest within the reviewed studies. Only one study comprehensively provided their eye-tracking data processing method to derive eye movement outcomes, as the article developed and validated an eye-tracker algorithm to derive saccades while walking in mTBI and controls , fixation (n = 3), smooth pursuit (n = 13), and nystagmus (n = 4) eye movements, and there were a plethora of outcomes reported for these eye movements  were generally impaired in mTBI compared to controls or baseline tests, regardless of acute or chronic mTBI status . Yet, thOther notable methodological limitations were found in the reviewed studies that may impact outcome interpretation. Studies examined the same eye movements but with slightly different protocols. For example, smooth pursuits were examined with a range of frequencies (0.1–1.25 Hz) and the visual stimulus  used for eye movement tasks varied across all studies . AlthougIn order to refine the information provided in our detailed tables , 2 a briThe majority of the reviewed studies that provided mTBI diagnostic criteria involved a clinician with experience of sports injuries.Included participants tended to be free of previous mTBI, musculoskeletal, cognitive, emotional, visual, or neurological issues.Eye-tracker sampling frequency of 500 Hz.Seated/static testing using computer screens to provide visual stimuliThe most common eye-tracker metrics were based upon the latency, gain, velocity, duration, or positional error rates of the specific eye movement assessments, with the velocity of eye movements being the most prominent outcome reported across studies.This review examined 22 studies that reported quantified eye movements in healthy controls and mTBI subjects. We reviewed; (i) how eye-movements were measured; (ii) reported eye-movement outcomes and their definitions; and (iii) differences reported between mTBI and controls in eye-movement outcomes. Across all of the reviewed studies there was a lack of basic methodological reporting, as there was little consensus or reporting of participant mTBI classification (acute/sub-acute or chronic), demographic characteristics  and inclusion or exclusion criteria. This limits the generalizability of results to mTBI populations and influences the reproducibility of the methods and results of the reviewed studies. Despite these limitations, this review has demonstrated that eye-movement measurement in mTBI is emerging, but further work is required to establish the validity and reliability of instrumentation and methods to derive eye-movement outcomes, as well as the nature of eye-movement impairments in mTBI.There is currently no “gold standard” instrument for eye-movement measurement, which is likely the reason for the numerous different instruments used in the reviewed studies. The majority of studies used static infra-red eye-tracking devices in constrained seated activities , but several studies did show progression to unconstrained dynamic eye-tracking protocols  mTBI cohorts and as a result the number of outcomes reported may lead to inappropriate statistical analysis or reporting due to the number of performed statistical comparisons . There is currently no universally accepted definition or diagnostic criteria for mTBI  to novel [e.g., eye skew (Howell et al., We were surprised that only two studies (Maruta et al., Previous eye-tracking studies have generally involved static seated eye-movement assessment tasks (Pelz and Canosa, Many previous studies of eye-movements in mTBI have incorporated subjective, clinical assessments that can be performed is any environment, such as the VOMS (Mucha et al., Aspects of eye-movements are impaired following an mTBI, as demonstrated by the reviewed studies that quantified impairments in either saccadic, smooth pursuit, fixation or nystagmus eye movements. While there is evidence from these studies, it is not yet strong enough to adopt quantitative eye-movement assessment within clinical practice. This review has highlighted that methodological issues across the current literature limit the understanding and generalizability of the reported findings in mTBI. There is a need for consensus on methods used and reporting of eye movement data, including eye-movement instruments, data collection procedures, processing algorithms and analysis methods are required. Comprehensive reporting of demographics, mTBI-related features, and confounding variables are also recommended for future work in this area . DevelopSS wrote the first draft and revisions of the manuscript. SS, LP, and DM designed the search strategy and conducted the literature search. SS and LK designed, implemented, and oversaw the topic area. SS, LP, DM, RP, JC, and LK were involved in interpretation of the data, writing the manuscript and revisions, final approval of the version to be published, and agree to be accountable for all aspects of the work.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.


The developing nervous system is a complex yet organized system of neurons, glial support cells, and extracellular matrix that arranges into an elegant, highly structured network. The extracellular and intracellular events that guide axons to their target locations have been well characterized in many regions of the developing nervous system. However, despite extensive work, we have a poor understanding of how axonal growth cones interact with surrounding glial cells to regulate network assembly. Glia-to-growth cone communication is either direct through cellular contacts or indirect through modulation of the local microenvironment via the secretion of factors or signaling molecules. Microglia, oligodendrocytes, astrocytes, Schwann cells, neural progenitor cells, and olfactory ensheathing cells have all been demonstrated to directly impact axon growth and guidance. Expanding our understanding of how different glial cell types directly interact with growing axons throughout neurodevelopment will inform basic and clinical neuroscientists. For example, identifying the key cellular players beyond the axonal growth cone itself may provide translational clues to develop therapeutic interventions to modulate neuron growth during development or regeneration following injury. This review will provide an overview of the current knowledge about glial involvement in development of the nervous system, specifically focusing on how glia directly interact with growing and maturing axons to influence neuronal connectivity. This focus will be applied to the clinically-relevant field of regeneration following spinal cord injury, highlighting how a better understanding of the roles of glia in neurodevelopment can inform strategies to improve axon regeneration after injury. Combinatorial actions of many well-characterized extracellular and intracellular events guide axons to their target locations, which is heavily influenced by tissue mechanics, bound and soluble secreted chemical factors, and cell-cell interactions. The interactions between axonal growth cones and surrounding cells within the developing nervous system is an important component of neurodevelopmental biology but is often not well characterized due to the challenges with observing these transient cellular interactions in vivo.The developing nervous system is a complex Glial cells in the developing nervous system have many supportive physiological functions, like maintaining solute and nutrient homeostasis, as well as migratory and synaptic development and maintenance roles, such as assisting neurons in reaching their target locations . Glial cThis review will provide a brief overview of the current knowledge about glial cell contributions to axon growth and guidance, specifically focusing on how glial cells directly interact with growing axons to influence neuronal connectivity. A selection of examples where glial cell-axonal growth cone interactions are shown to play a crucial role during neurodevelopment will be critically discussed. This basic science background is then linked with translational work targeting glia to promote regeneration following spinal cord injury, highlighting how a better understanding of the role of glia in neurodevelopment can inform strategies to improve axon regeneration after injury.Glial cell development in the central nervous system (CNS) and peripheral nervous system (PNS) often occurs alongside neuron development and maturation as many glial subtypes originate from a common precursor stem cell . TherefoMicroglia are the only glial cell to enter the CNS from the periphery, doing so well before CNS-resident glia differentiate . Unlike Astrocytes and oligodendrocytes, the macroglia in the CNS, originate from a common precursor, the radial glial (RG) cell . RG cellIn the PNS, glial cells are derived from neural crest cells that differentiate while migrating to their final destination . These mFinally, olfactory ensheathing cells (OECs) are a unique population of Schwann cells that facilitate the replenishment of olfactory neurons . This unDrosophila early embryogenesis, three classes of glial cells form an organized pattern at each body segment before axon outgrowth occurs, and these cells enwrap the axon tracts as they migrate  of the astrocytes in the culture, which exhibited a variety of shapes and arrangements, and often co-localized with oligodendrocytes. The atypical astrocytes exhibited a spindle shape with a high cell density arranged in a polarized fashion and covered by fewer oligodendrocytes. When dorsal root ganglia (DRG) neurons were plated on top of this heterogeneous astrocyte population, the typical astrocytes promoted neuron adhesions and neurite growth consistent with prior studies  and may be related to damaged or reactive astrocytes that have a well-characterized inhibitory effect on neurite growth both in vitro and in vivo . Clearly an important control experiment is to determine if these findings are reproducible in a rodent model with selective astrocyte ablation, which has been generated in other laboratories  are notorious for their inhibitory effect on axon growth and guidance . Early sIn vitro studies on Nogo ligands suggest these inhibitory molecules expressed on oligodendrocytes and myelin may serve to prevent axon sprouting in the adult CNS  integral membrane proteoglycan expressed by OPCs during neurodevelopment . NG2 belll types  and perill types , this raquestion .Microglia are CNS-resident macrophages that serve a number of important roles in regulating tissue homeostasis, namely phagocytic scavenging, localized immune function, modulation of synaptic transmission, synaptogenesis, and neurotrophic support . Microgl+ axon fragments within their cytosol, suggesting a phagocytic role. Interestingly, microglial depletion resulted in enhanced growth of the dopaminergic axons within the subpallium, while maternal excess immune activation of microglia resulted in reduced axon growth. Similarly, microglia also play a role in localization of LIM/homeobox 6 (Lhx6)-positive interneurons, which originate in the subpallium, migrating tangentially into the neocortex and eventually migrating radially into the cortical plate. However, when microglia were depleted, Lhx6-interneurons entered prematurely into the cortical plate and had more diffuse localization in layer V, which persisted postnatally  inhibited neuron outgrowth and induced growth cone collapse (repulsive guidance molecule a (RGMa), a glycosylphosphatidylinositol (GPI)-linked glycoprotein that has been previously demonstrated to induce growth cone collapse of retinal axons , a membrane-bound, cleavable chemorepellent found on the OEC cell surface, was found to mediate this defasciculating and sorting process as olfactory neuron axons avoided regions with high Sema3A expression. Furthermore, Sema3A-deficient mice exhibited defects in olfactory neuron axon sorting within the olfactory nerve layer, which persisted into postnatal life (Olfactory neurons are unique as they are continuously turned over so new axons must enter the CNS from the periphery throughout life. Olfactory neuron axons are supported by olfactory ensheathing cells (OECs) in both the periphery and within the olfactory bulb, which both aid in the growth and guidance of olfactory axons as well as surround groups of olfactory axons to enhance electrical conduction . Cell suthe OECs . Interesthe OECs . Studies propria . These f propria . When prtal life . Overallin vivo imaging is necessary to elucidate these details in neurodevelopment.Beyond astrocytes, microglia, and OECs, there is little evidence that Schwann cells or Schwann cell precursors (SCPs) play a direct role in axon growth and guidance during PNS development. Rather, the functions of SCPs during neurodevelopment are believed to include trophic support of sensory and motor axons and nerve myelination . Early sMultiple families of cell adhesion molecules (CAMs) mediate interactions between glial cells and neurons, with some being specific for certain glial subtypes see . Many ofcis or trans that allow for a variety of functions depending on differential expression and binding interactions of the cytoplasmic tail (reviewed in The immunoglobulin superfamily (IgSF) of CAMs, including L1-CAM family members NCAM and CHL1, as well as N-cadherin, mediate much of the attractive interactions between glial cells and axonal growth cones . The L1-trans. The intracellular cytoplasmic tail of cadherins associate with p120 catenin complexes to link them to the actin cytoskeleton , which belongs to the larger family of calcium-dependent adhesion molecules, promotes cell adhesion through hemophilic binding in The inhibitory or repulsive interactions between glial cells and axon growth cones include the myelin-associated inhibitors (MAIs), semaphorins, NG2, and RGMa . The oliin trans to promote the formation of neogenin receptor dimers, which initiates downstream signal transduction. The co-receptor Unc5B, a member of the netrin family, was found to interact with neogenin, which activates downstream signaling to the leukemia-associated Rho GEF (LARG)  signal through the RhoA/ROCK pathway to inhibit axon growth and guidance. RGMa is part of the larger family of GPI-linked repulsive guidance molecules (RGMs) that signals through interactions with the type 1 transmembrane protein neogenin (reviewed in F (LARG) . RGMa-inF (LARG) . Thus, LF (LARG) . AdditioF (LARG) . NG2 alsF (LARG) . AdditioF (LARG) .Semaphorins, a large family that includes GPI-linked and membrane-bound proteins that play critical roles in axon growth and guidance, signal through both RhoA-dependent and independent pathways  . This seIn an injured state, cytokines, cell fragments, and nucleic acids contribute to differentiation of CNS microglia into “classically activated” M1 or “alternatively activated” M2 subtypes . This de–/–), the microglial-specific fractalkine chemokine receptor, improved axonal growth of dopaminergic neurons in the subpallium  and LPS . Additionally, many other pre-clinical studies have demonstrated the beneficial effects of the RGMa-suppressing antibiotic minocycline in SCI  with mixed results, often challenged by small sample sizes and technical difficulties with cell extraction and transplantation (In addition to the identification of novel drug targets, pre-clinical studies investigating glial-axon growth cone interactions have led to the notion of implanting growth-promoting glia into the site of injury. For example, OECs can be harvested from the olfactory blub, cultured  animals . OEC tra animals . Interesantation . There aantation . Therefoin vivo, much of these developmental events will become accessible to view in real time, which could promise a plethora of important information on glial-axon growth cone interactions (In the CNS and PNS, many glial cell types are able to affect axon growth and guidance, which has an impact on neuronal wiring in adulthood as well as the outcomes of several disease states, such as SCI see . Promisiractions . Furtherractions , as wellractions . Finallyractions . In the MR wrote the manuscript. TG and LP revised the manuscript.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.


Infection with hepatitis B virus (HBV) remains a global health challenge. Approximately 292 million people worldwide are chronically infected with HBV and the annual mortality from the infection is approaching 900,000. Despite the availability of an effective prophylactic vaccine, millions of individuals are at risk of potentially fatal complicating cirrhosis and hepatocellular carcinoma. Current drug treatments can suppress viral replication, slow the progression of liver fibrosis, and reduce infectivity, but can rarely clear the viral covalently closed circular DNA (cccDNA) that is responsible for HBV persistence. Alternative therapeutic strategies, including those based on viral gene silencing by harnessing the RNA interference (RNAi) pathway, effectively suppress HBV replication and thus hold promise. RNAi-based silencing of certain viral genes may even lead to disabling of cccDNA during chronic infection. This review summarizes different RNAi activators that have been tested against HBV, the advances with vectors used to deliver artificial potentially therapeutic RNAi sequences to the liver, and the current status of preclinical and clinical investigation. Problems arising as a result of chronic infection with hepatitis B virus (HBV) continue to pose major global health challenges (reviewed in ). AnnualThe virion of HBV, or Dane particle, has large, middle, and small surface antigens (HBsAgs) embedded in a surrounding envelope (reviewed in ). The nuAfter traversing the cell membrane, the nucleocapsid is released and transferred to the nucleus  Figure . This prcccDNA is a stable replication intermediate and serves as the template for transcription of pregenomic RNA (pgRNA) and viral protein-encoding mRNAs . MaintenHBV transcripts are initiated at the basic core promoter/enhancer II, PreS1, S, and X promoters (reviewed in ). HoweveVarious types of cure from HBV infection have been defined . A functThe rationale for treating infection with HBV is to minimize or eliminate complications that result from persistence of the virus. Currently there are two main classes of drug available to treat HBV infection: nucleoside/nucleotide analogues (NAs), which disrupt viral DNA synthesis during reverse transcription, and interferon-alpha (IFN-α) (reviewed in ). The siA variety of new approaches is being developed for treatment of chronic infection with HBV. Gene editing has gained popularity and is based on targeted mutation of HBV sequences, particularly cccDNA (reviewed in ). Use ofHBx means that different viral transcripts may be targeted by individual silencing sequences  is a ubiquitous post-transcriptional gene silencing (PTGS) mechanism found in metazoan cells. Potent and specific gene silencing is affected by small duplex RNAs such as 21–23 nt short interfering RNAs (siRNAs) and ≈22 nt microRNAs (miRNAs). In mammalian cells, miRNAs play a fundamental role in gene regulation and are generated by the miRNA biogenesis pathway (reviewed in ). AlthouGenes encoding miRNAs are transcribed to produce precursor transcripts from which the mature sequences are generated in a stepwise manner that involves action of RNase III enzymes (reviewed in ). CharacOne strand of the miRNA duplex is selected as the mature miRNA or guide sequence and loaded onto an Argonaute (Ago) protein of the RNA-induced silencing complex (RISC) (reviewed in ). Prior The miRNA typically directs RISC to partially complementary binding sites in the 3′ untranslated region (UTR) of messenger RNAs (mRNAs), which are then targeted for translational repression, deadenylation, and degradation (reviewed in ). AlternIt is ironic that exploitation of the RNAi pathway preceded its complete elucidation. Following the demonstration by Fire and colleagues that introduction of long double-stranded RNA into the nematode worm caused potent and specific silencing , numerouUse of DNA to express RNAi activators was also explored at an early stage. This entailed expression of each individual strand of an siRNA from two expression cassettes  or the tThe discovery that RNAi could indeed be triggered in mammalian cells by synthetic siRNA  led to aThe designs of synthetic siRNAs were such that the structural features did not deviate significantly from those established in earlier studies  and invaIncorporation of altritol, a six-membered ring in place of ribose, into synthetic anti-HBV siRNA improved silencing activity but at a cost of some hepatoxicity in vivo . In contLys promoter [Although chemical modification to siRNAs may increase their half-lives to prolong therapeutic silencing, repeated dosing is required to treat chronic HBV infection effectively. Renewable expression of RNAi activators from DNA cassettes has the potential for more durable silencing. RNA polymerase III-driven transcription, which naturally produces short defined RNA sequences, was initially explored for expression of RNAi activators . Transcrpromoter  also silpromoter ,58. In ppromoter ,60. Thispromoter  and adenpromoter  to delivHBV is considered a good target for potential treatment on the basis of RNAi ,59 but sIn a study aimed at evaluating efficacy of expressed shRNAs against genotypes A-H, the researchers designed 21 shRNAs to target conserved regions . Six of To advance RNAi-based therapy for HBV infection, use of models that accurately simulate HBV infection and replication is important. Improved understanding of the molecular biology of HBV replication has been beneficial, but suitability and convenience of models that reproduce all stages of HBV infection remain a concern. A factor that has impeded progress was incomplete understanding of the mechanism of HBV entry into hepatocytes. Until fairly recently, cultured cells that could be infected with the virus were limited to primary human hepatocytes (reviewed in ) and theTupaia belangeri) can be infected by HBV, but these animals have not yet been widely used for evaluation of RNAi-based treatment of HBV infection [Preclinical studies in mice have commonly relied on plasmid-derived ,57,58 ornfection .Chimpanzees are the only primates susceptible to HBV infection and are an excellent model of chronic HBV infection. However restrictions on use of chimpanzees, high costs, and ethical concerns have limited their use as a preclinical model . AlternaAn important goal in the translation of RNAi-based therapies for HBV is targeted delivery to the liver. Several delivery strategies have been investigated, which include synthetic non-viral formulations and recombinant viral vectors, which are used for delivery of synthetic and expressed RNAi activators, respectively.Lipid nanoparticles (LNPs) were used in a number of earlier studies to deliver siRNAs (reviewed in ). These X also effectively silenced HBV replication [In an early study, chemically modified siRNAs targeting HBV were incorporated into stable nucleic acid lipid particles (SNALPs) then administered by intravenous injection to mice replicating HBV . This SNlication .N-acetylgalactosamine  have become a popular choice for liver-targeted delivery. Conjugation of siRNAs to ligands derived from GalNAc enables uptake by the asialoglycoprotein receptor (ASGPR) on hepatocytes, resulting in liver-specific delivery of siRNAs in vitro and in vivo [More recently, conjugate-mediated delivery of siRNAs has gained momentum in preclinical and clinical applications. siRNAs conjugated to  in vivo . A singl in vivo . A simil in vivo ,97, but In a recent advancement, GalNAc was modified to produce a triantennary GalNAc ligand that enabled robust and durable gene silencing in the liver following subcutaneous administration to mice . In furtViruses have evolved efficient mechanisms of cell transduction, and this feature has been exploited to make viral vectors that have been used successfully to deliver expressed gene silencers in vivo (reviewed in ). TypicaRecombinant lentiviral vectors (LVs), adenoviral vectors, and adeno-associated viral vectors (AAVs) have all shown good transduction efficiency of the liver, and have therefore been a logical choice for delivery of anti-HBV sequences ,60,61,62AAVs have emerged as good candidates for delivery of gene-based therapies. Initial concerns about oncogenic potential of AAVs  were all11 vector genome copies per mouse, which was a 10-fold lower dose than that which was reported in a previous study [Use of AAVs to deliver anti-HBV shRNAs or miRNAs has been extensively explored ,110,113.us study . AAV-basus study .Despite their impressive efficacy and safety profile, rAAVs are not without shortcomings. High prevalence of pre-existing immunity to vectors derived from popular serotypes, such as AAV2 or AAV8, leads to short-lived transgene expression in humans ,117. ThiIn silico construction of synthetic AAV vectors has been a particularly promising approach to avoiding pre-existing immunity and has been used to generate ancestral genes encoding synthetic AAV capsid variants ,121. TheUse of siRNAs has reached clinical evaluation for numerous diseases, including chronic HBV infection. GalNAc-conjugated siRNAs developed in preclinical studies by Arrowhead Pharmaceuticals ,97 have 10 IU/mL in all 40 patients and HBsAg < 100 IU/mL in 88% of patients over 24 weeks. Importantly, all measurable viral products were reduced in both HBeAg-positive and -negative patients. JNJ-3989 is currently being investigated in a phase II clinical trials in combination with a nucleoside/nucleotide analogue and JNJ-6379, a capsid assembly modulator .These studies showed the previously underappreciated contribution of HBsAg expression from integrated copies of the HBV genome, as opposed to cccDNA, and highlighted the need for additional siRNAs capable of targeting all HBV transcripts, regardless of origin. A second-generation siRNA targeting all HBV transcripts, ARC-521, was found to reduce HBsAg and HBV DNA levels in a phase I trial (NCT02797522). However, both ARC-520 and ARC-521 trials were discontinued following the observation of lethal toxicity of the EX1 delivery formulation, a version of NAG-MLP, in a safety study in non-human primates. Subsequently, a GalNac-conjugated siRNA capable of targeting all HBV transcripts  and administered subcutaneously is under investigation (NCT03365947) and being developed in collaboration with Janssen Pharmaceuticals. Clinical data released thus far indicates that JNJ-3989 is well-tolerated in patients with CHB, with a HBsAg reduction by ≥1 log10 was observed in all 12 patients (https://investor.arbutusbio.com/node/13611/pdf). Despite encouraging results, the development of ARB-1467 has been discontinued. In an ongoing phase I/II study by Vir Biotechnology in collaboration with Alnylam Pharmaceuticals, the VIR-2218 (formerly ALN-HBV02) GalNAc-conjugated siRNA is being assessed in healthy and chronically infected volunteers to determine safety, tolerability, and antiviral efficacy (NCT03672188). Vir Biotechnology has indicated that preliminary results are promising with a durable, dose-dependent reduction in HBsAg, but official data have not yet been published.A siRNA delivered intravenously using LNPs (ARB-1467) was also assessed in a phase II clinical trial by Arbutus Biopharma (NCT02631096) . The treOver the past 20 years, synthetic siRNAs and expressed activators of the RNAi pathway have proved to be capable of silencing HBV replication in vitro and in vivo. Durability of silencing efficacy achieved by expressed HBV-targeting sequences, such as those that are encoded by apri-miRNA expression cassettes delivered with viral vectors, is a very useful property for treating the chronic disease. However, advancing the technology to enable inexpensive production of viral vectors, avoidance of host immunity, and efficient transduction of hepatocytes need to be tackled successfully before this approach is translated to a clinical setting. Synthetic anti-HBV siRNAs in non-viral formulations are currently at a more advanced stage than HBV-targeting viral vectors, and are being tested in clinical trials. Available clinical data are promising and indicate that anti-HBV siRNAs are safe and well-tolerated. Given the large number of people who are chronically infected with HBV, an important potential advantage of these synthetic drug candidates is that they are more conveniently amenable to scalable and economical production.HBx have a particularly suppressive effect on transcription from cccDNA. Inhibition of transcription from cccDNA, especially if it is sustained, will be a significant achievement and go some way to achieving a functional cure for HBV infection. An added benefit of directing RNAi activators to HBx is that the sequence is common to all viral transcripts, and inhibition of expression of all viral proteins may be achieved by targeting this gene.Progress with optimizing effectors of gene silencing and identifying good HBV targets has been impressive. Silencers have been directed to sequences that encompass almost the entire HBV genome, but particularly good efficacy does not appear to result from targeting specific viral sequences. Nevertheless, certain viral targets may become more favored for reasons that are not yet completely understood. Improved knowledge about the cellular mechanisms of HBV infection and replication, particularly as they pertain to cccDNA biogenesis, transcription, and degradation, may provide the insights that are necessary to improve selection of viral cognates. HBx has recently been shown to facilitate degradation of the structural maintenance of chromosomes (SMC) 5/6 complex ,124. TheDeveloping new approaches to economic preparation of vectors, avoidance of host immunity, characterization of pharmacokinetics, exclusion of unintended off-target silencing, and assay of biomarkers of cccDNA in carriers are current priorities. Moreover, available animal models of HBV infection have limitations, and testing candidate drugs in settings that closely simulate natural chronic HBV infection will facilitate drug development. Meeting these goals will provide an impetus. Nevertheless, overall progress in the field indicates that potent, effective silencing of viral transcripts generated from cccDNA in chronic HBV infection is achievable and has therapeutic potential. Results from current and future clinical trials are keenly awaited, and outcomes from these studies will pave the way for achieving the goal of a functional cure for chronic HBV infection.


Infectious disease of poultry and pig are major threat to health and cause severe economic loss to the food industry and a global food safety issue. Poultry and pig act as a mixing vessel of zoonotic transmission of disease to humans. Effective mucosal vaccines used in animals could reduce the impact of diseases in food animals. Chitosan is a biocompatible polymer, and its positive charge makes it a natural mucoadhesive agent. Therefore, since last one-decade chitosan derived nanoparticles (CS NPs) have been in use widely to deliver vaccine antigens in animals through mucosal route. Primary route of entry of most infectious disease pathogen is through oral and nasal routes, and the CS NPs based vaccines delivered through that routes enhance the immunogenicity of encapsulated vaccine antigens by targeting the cargo to mucosal microfold cells, dendritic cells and macrophages. Resulting in induction of robust secretory and systemic antibodies and/or cell mediated immune response which provides protection against infections. To date, CS NPs is being widely used for mucosal vaccine delivery in poultry and pigs to control bacterial and viral infections, and tested in several preclinical trials for vaccine delivery in humans. In this review, we highlighted the progress so far made in using CS NPs as a vehicle for mucosal vaccine delivery against infectious and zoonotic diseases of poultry and pigs. Discussed about the need of CS NPs modifications, CS NPs based vaccines induced immune responses and its role in protection, and challenges in vaccination and future directions. Traditionally vaccination is the preferred and effective approach to control and prevent infectious diseases . The innD-glucosamine and N-acetyl-D-glucosamine , Polyanhydride, Sodium alginate, Starch, and Chitosan. Among them chitosan is widely used to target mucosal sites by oral, ocular, nasal, implant, parenteral, and transdermal administration due to its unique properties such as positive surface charge and flexibility to do modification and conjugation with other polymers . The CS + T helper cells which interact with B cells to generate IgA committed cells (IgA+ B cells). The IgA+ B cells migrate to effector sites, differentiate into IgA producing plasma cells and secrete IgA, leading to induction of mucosal immune response ; Mucosal immune system consists of mucosa-associated lymphoid tissues (MALT), present along the mucosa of the respiratory tract and gastrointestinal tract as NALT and GALT . The oraresponse , 4. Whilresponse , 4. HencSalmonella, Campylobacter, toxin-producing strains of Escherichia coli and Listeria monocytogenes, and zoonotic transmission of the latter even cause death in humans  and Newcastle disease virus (NDV) to mucosal sites in poultry  is a major economic threat to poultry industry causing food-borne illness in humans  and flagellin protein entrapped and surface flagellin protein coated CS NPs (nanovaccine) delivered orally in layer chickens induced antigen specific IgA response in cloaca but did not elicit the IgG antibody response in serum. In nanovaccine inoculated Salmonella challenged birds observed enhanced anamnestic specific IgA response in cloaca, ileum and bile samples, with increased serum IFN-γ level and antigen specific lymphocyte proliferation responses were observed. Salmonella nanovaccine immunization also increased the Toll-like receptor (TLRs)-2 and -4, IFN-γ, TGF-ß, and IL-4 gene expression. Although due to high bacterial challenge dose [1 × 109 colony-forming unit (CFU) per bird] used in that nanovaccine trial we did not detect substantial reduction in the bacterial colonization, but our this first study confirmed that surface flagellin protein coating on CS NPs is necessary to reach the vaccine to ileum immune cells of layer chickens when delivered orally  infection. Oral gavage and drinking water delivered nanovaccine immunization induced immune response correlated with reduction in Salmonella load in cecum  inoculation only increased different TLRs mRNA expression and specific splenocytes proliferation, but did not induce high levels of antigen specific antibody response . Likewisresponse . Whereashe cecum . Mannose vaccine . AltogetCampylobacter jejuni (C. jejuni) is a helical-shaped, non-spore forming; Gram-negative enteric bacterium belongs to Campylobacteraceae family. It is a most common food borne pathogen globally. Poultry is the natural host for C. jejuni and contaminated meat is the main source for human infection  stay in the intestines and causes disease in other organs and systemically  delivered intranasal in chickens induced anamnestic haemagglutination inhibition (HI) titers and mucosal IgA response. In the field studies, CS NPs vaccination efficiently controlled AIV outbreaks in chicken with high HI antibody titer, rescued the loss in egg production without infection, while the commercial intramuscular inactivated AIV vaccine failed to provide protection. This study suggests that combination of intranasal vaccine with commercial intramuscular vaccine is more comprehensive as a prophylaxis against the disease, and they used chitosan instead of CS NPs , and it belongs to the family Arteriviridae . Pig is Poultry and pigs are susceptible to many bacterial and viral infections. Experimental vaccine trials conducted in a natural host of disease is more appropriate than in rodent models as they lead to quick translation of technology to make a commercial product. Pig and poultry may serve as an appropriate animal models to study performance of respiratory and enteric vaccines, respectively. Pig anatomy, genetics and lung structure and physiology are more similar to humans, and thus pig is considered as a useful biomedical animal model for human respiratory diseases. The CS NPs vaccine delivery platform has been used efficiently to administer some of the viral and bacterial vaccines to mucosal sites of pigs and poultry, and still there is a scope to evaluate CS NPs platform for many other infectious and zoonotic diseases. The CS NPs vaccine delivery strategy used to mitigate infectious diseases in poultry and pigs showed benefits with induction of robust mucosal secretory IgA antibody and variable levels of systemic antibodies, cytokine production and cell mediated immune response. Further studies are required to improve efficiency of the vaccine through innovative modifications to the CS NPs vaccine technology. This include application of suitable surface modifications in CS NPs, optimizing its size, shape and surface charge depending on the route of delivery. Further, to achieve increased breadth of immunity and balanced Th1-Th2 immune response for superior cross-protection against many viral and bacterial diseases the following parameters are important: (i) type of antigen and its concentration, (ii) secondary adjuvant/s, (iii) age of animal at the time of prime vaccination, (iv) number of doses, and (v) route of vaccination.SR conceived and wrote the manuscript. GR edited and revised the manuscript. Both authors read and agreed the manuscript 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.


Upper airway injury secondary to blunt neck trauma can lead to upper airway obstruction and potentially cause a life-threatening condition. The most important aspect in the care of laryngeal trauma is to establish a secure airway. Focused airway ultrasound enables recognition of important upper airway structures, offers early opportunity to identify life-threatening upper airway injury, and allows assessment of the extent of injury. This information that can be obtained rapidly at the bedside has the potential to facilitate rapid intervention.We report a case series that illustrate the diagnostic value of focused airway ultrasound in the diagnosis of laryngeal trauma in patients presenting with blunt neck injury.Early recognition, appropriate triaging, accurate airway evaluation, and prompt management of such injuries are essential. In this case series, we introduce the potential role of focused airway ultrasound in suspected laryngeal trauma, and the correlation of these exam findings with that of computed tomography (CT) scanning, based on the Schaefer classification of laryngeal injury. Laryngeal injuries are often undiagnosed in the initial evaluation of the trauma patient. They are rare, with an estimated incidence of one in every 30,000 emergency department admissions . DelayedBlunt laryngeal trauma may present with varying degrees of severity, from mild to life-threatening extremes. A tracheostomy may be required to gain airway access distal to the site of injury. A systematic classification and management approach of blunt laryngeal trauma is crucial to guide early decision-making and improve patient outcome in the emergency department.Current standard of care for laryngeal trauma is determined according to the Schaefer classification of laryngeal injury. Schaefer group 1 and group 2 with minor endolaryngeal injuries can be managed conservatively with observation, antibiotics, steroids, voice rest and anti-reflux medications. However, for more severe Schaefer type 3–5 injuries, open surgical repair will be required to secure a definitive airway [Good history taking, detailed clinical examination and a high index of suspicion are critical in the diagnosis of laryngeal trauma. The diagnosis can be aided using flexible nasendoscopy by direct visualization of the airway. CT scanning of the neck is still considered the gold standard to grade the severity of the injury and to direct appropriate management. Obtaining a timely CT scan may often be challenging due to logistical problems, primarily availability of radiology service support especially in resource-limited area, and stability for patient transfer.Of late, studies have integrated the use of upper airway ultrasound into point-of-care ultrasound examination, a paradigm shift in upper airway assessment –5. The iWe discuss the role of focused airway ultrasound in upper airway trauma performed by point-of-care ultrasound trained emergency physician and propose a focused airway ultrasound classification in relation to Schaefer classification of laryngeal injury.A 24-year-old male presented with neck swelling without signs of respiratory distress after a traumatic blunt neck injury. There was swelling of the anterior neck without palpable crepitus. Airway ultrasound showed disruption of the air–mucosal interface suggesting endolaryngeal disruption Fig. b. CT scaA 66-year-old motorcyclist, was injured in a collision with a van. He presented with mild neck pain, difficulty in breathing, hoarseness, dysphagia and odynophagia. He had stridor, and his neck was swollen and tender with subcutaneous emphysema.Airway ultrasound using a 15-MHz linear transducer found discontinuity of the anterior cortex of thyroid cartilage with minimal surrounding tissue edema, consistent with Schaefer group 2 . Further study is needed to determine if focused upper airway ultrasound can reliably be used to determine injury grade [While the authors could not find previous publication of ultrasound assessment for upper airway injury in trauma, prior research by Osman et al. and You-Ten et al. briefly illustrated the usage of airway ultrasound in a step-by step manner to delineate the normal sonoanatomy of the upper airway such as thyroid cartilage, epiglottis, cricoid cartilage, cricothyroid membrane, tracheal cartilages, esophagus and the surrounding soft tissues –5. SchicCheng et al.  found goUpper airway ultrasound findings that correlate with the Schaefer Classification System may be especially relevant in hemodynamically unstable patients where CT imaging is not feasible. While larger trials are needed, we propose that focused airway ultrasound can be used to correlate with the Schaefer Classification System Table  and therIn centers without CT scan capabilities—focused airway ultrasound can complement emergency department triage protocol to enable early airway management planning in blunt laryngeal injury.In centers with CT scan facility—focused airway ultrasound can hasten airway management planning prior to airway catastrophe during an emergency situation when transfer to the radiology suite is deemed unsuitable.In resource-limited situation such as—scarce resources, remote area, and humanitarian medical mission in environmental disasters and war-torn regions—focused airway ultrasound can supplement disaster and transfer protocol facilitating decision for early airway intervention while preparing for emergency transfer.It has the added advantage of real-time visualization of dynamic vocal cords function.Possible advantages of focused upper airway ultrasound in the diagnostic classification of blunt laryngeal trauma are:Ultrasound evaluation of the airway may not be practical in every case. Subcutaneous emphysema, posterior laryngeal injury, cartilage calcification and foreign bodies may result in artifacts interfering with ultrasound images and interpretation. Furthermore, ultrasound techniques and interpretation are operator-dependent, and have a steep learning curve. Adequate competency training and reproducibility is important to standardize findings.Validation studies comparing accuracy of focused airway ultrasound to CT scan findings across a range of injury types.Management outcome, cost effectiveness, accuracy and time of diagnosis of focused airway ultrasound compared to existing radiological modalities.Reproducibility of results by different operators at different stages of seniority and proficiency level, their abilities to accurately detect pathology and studies on learning curve of this procedure.Further studies to identify optimal management strategies for patients with laryngeal injury are required. Areas of interest include:Ultrasound assessment of the upper airway is a promising adjunct in the rapid evaluation of patients with suspected laryngeal trauma. Early diagnosis and injury classification stratification with point-of-care ultrasound may play an important role in trauma patient care, particularly those too unstable for CT imaging or when advanced imaging is unavailable.Additional file 1: Video S1. Bedside airway ultrasound revealed a displaced fracture of the thyroid cartilage, disruption of anterior cortex of thyroid cartilage with surrounding mixed echogenicity denoting endolaryngeal edema.


The ideal situation would, however, be to carry out well-designed clinical trials in humans  using adequate quantities of the suspected probiotic molecule(s) or adequate quantities of isogenic knock-out or knock-in probiotic mutants. In this review, we discuss our current knowledge about probiotic bacteria and yeast molecules that are involved in molecular signaling with the host. We also discuss the challenges and future perspectives in the search for probiotic effector molecules.Understanding the mechanism behind probiotic action will enable a rational selection of probiotics, increase the chances of success in clinical studies and make it easy to substantiate health claims. However, most probiotic studies over the years have rather focused on the effects of probiotics in health and disease, whereas little is known about the specific molecules that trigger effects in hosts. This makes it difficult to describe the detailed mechanism by which a given probiotic functions. Probiotics communicate with their hosts through molecular signaling. Meanwhile, since the molecules produced by probiotics under  Probiotics are live microorganisms which when administered in adequate quantities provide a beneficial effect to the host . Over thLactobacillus rhamnosus GG has been shown to secrete protein p40, which stimulates ADAM17 activation and heparin binding-epidermal growth factor (HB-EGF) release. 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Dopaminerotonin  by influerotonin , it is nerotonin .Lactococcus lactis for instance produces nisin (a bacteriocin), which can permeate the cell membrane of pathogenic Gram positive-bacteria and bind to Lipid II to inhibit cell wall synthesis  1 and 2 replication  and invH (a Staphylococcus typhimirium invasion lipoprotein gene) thereby reducing the ability of these pathogens to attach to the host cells  protein synthesis and viral replication  using KduI and KduD enzymes  in macrophages (L. acidophilus A4 to reduce serum cholesterol. Wild L. acidophilus A4 strains effectively reduced serum cholesterol levels while catabolite control protein A (ccpA) mutants had significantly reduced cholesterol reducing abilities. Although the direct effect of the protein on cholesterol reduction remains unclear, the ccpA gene is known to strongly regulate important cell functions including lipid metabolism, cell envelope biogenesis, carbohydrate transport and metabolism, outer membrane and intracellular cargo trafficking . Such studies may also have to consider the influence of disease conditions on the immune system, host antimicrobial proteins, and gut microbial competition on the probiotic in other to ascertain what genes (and bioactive compounds) are really triggered (and produced) in consumed probiotics. A more plausible way of assessing the probable effects of these stressors on a consumed probiotic would be to collect the microbe from gut samples (or vagina) after the expected physiological effect has occurred and subjected to transcriptomics to ascertain if the mRNA of the suspected bioactive molecule (in case of a protein or peptide) is actively transcribed under those conditions.Over the years, server studies have successfully identified as soluble molecules of probiotics in media by using metabolomics approaches . After r effects . For mem effects  and test effects , and so acterium . TherefoFor a better understanding of which probiotic molecules trigger a given effect, there is the need to carry out well-designed clinical trials in humans using adequate quantities of the isolated bioactive molecules from probiotics or adequate quantities of isogenic knock-out or knock-in probiotic mutants. This is essential because animals and humans are different and so extrapolating results from animal studies may not always be correct. Meanwhile, such a study would face several ethical and technical hurdles as it involves humans and will have to establish the probiotic strain’s potency, effective dose, targeted host response, targeted host site, and other important parameters.ED conceived, designed, and wrote the manuscript. FO, CX, and RC revised and made corrections. D-HO approved the manuscript and provided funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.