Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was originally characterised as a novel respiratory coronavirus and was thought to primarily target pulmonary tissues in infected patients, similar to its close relative SARS-CoV, which was responsible for the epidemic of SARS in 2003. This preconception turned out to be an underestimation. Although SARS-CoV-2 does indeed infect pulmonary epithelial cells, it might also infect many other cell types, causing systematic inflammation with cytokine release and affecting multiple critical organs besides the lungs in severe cases.The Lancet. In another study, published in The Lancet Respiratory Medicine on February 17, researchers observed interstitial mononuclear inflammatory infiltrates in the heart tissue of a deceased patient with COVID-19. Furthermore, myocardial damage and heart failure have been reported to contribute to causes of death that were linked to COVID-19 complications. In addition to inducing an overreactive inflammatory response, recent studies have shown that SARS-CoV-2 might also directly attack vascular endothelial cells and disrupt vascular barrier, leading to disseminated intravascular coagulation and inflammatory cell infiltration. As our understanding of the disease pathology improves, evidence is emerging that vascular pathology could have a substantial role in COVID-19 disease outcome.In some patients, SARS-CoV-2 appears to attack the cardiovascular system, causing numerous cardiovascular complications. Back in January 2020, clinicians from Wuhan  reported myocardial injury in patients with COVID-19 in a study published by Lancet paper published on April 20, 2020, Frank Ruschitzka and colleagues from University Hospital Zürich  observed direct SARS-CoV-2 infection of endothelial cells and diffuse endothelial inflammation in vascular beds of different organs in patients with COVID-19. Indeed, the angiotensin converting enzyme 2 (ACE2) receptor required for SARS-CoV-2 infection is expressed on the surface of endothelial cells. Shortly after their study was published, several other post-mortem studies showed similar patterns of vascular damage in deceased patients who had COVID-19. For example, two studies published in The New England Journal of Medicine on May 21 and The Lancet Respiratory Medicine on May 27 showed distinctive vascular features of severe endothelial injury, widespread thrombosis with microangiopathy, and increased vascular angiogenesis in the lungs of patients with COVID-19. Thrombosis not only occurs in the infected lung, but also in other organs including the heart and kidneys, as Amy Rapkiewicz and colleagues reported in EClinicalMedicine on June 25. All these data indicate that vasculopathy is likely to be important in COVID-19 pathogenesis and endothelial cells could themselves have a role in orchestrating the destructive intravascular coagulopathy associated with SARS-CoV-2 infection.In a The Lancet Haematology on June 30, George Goshua and colleagues from Yale University  determined the role of endotheliopathy in COVID-19-associated coagulopathy pathogenesis and provided novel mechanistic insights into COVID-19-associated endotheliopathy. The authors discovered increased concentrations of plasma von Willebrand factor (VWF) in patients with COVID-19, which increased with disease severity. Plasma concentrations of soluble thrombomodulin correlated with clinical outcomes, with in-hospital mortality significantly lower in patients with low soluble thrombomodulin than in patients with high soluble thrombomodulin. Only endothelial cells and megakaryocytes can produce VWF, which has a major role in blood coagulation. As commented by O'Sullivan and colleagues in the same issue, these data support a mechanistic model in which alterations of plasma VWF and thrombomodulin concentrations following endothelial cell injury caused by SARS-CoV-2 infection lead to clinical prothrombotic manifestations of coagulopathy in patients with COVID-19. In other words, released VWF binds to platelets, neutrophils, and monocytes to initiate microvascular thrombosis; meanwhile, thrombomodulin further promotes a procoagulant and proinflammatory local milieu within the injured vasculature.Immune mechanisms have been proposed to explain COVID-19-associated intravascular coagulopathy. Injured endothelial cells cause vascular leakage, trigger uncontrolled blood clotting, and recruit different types of immune cells and immunological factors that result in widespread inflammation and further vascular damage, forming a vicious cycle. In a single-centre, cross-sectional study published in An increasing appreciation of the role of endothelial cells in COVID-19 pathogenesis has prompted research into vascular normalisation and anticoagulation strategies. For instance, bevacizumab, a monoclonal antibody targeting VEGF, can inhibit its vessel-permeabilizing activity and could help to maintain vasculature integrity in patients with COVID-19. Clinical trials  are exploring the effect of targeting this vascular factor on COVID-19 disease. One Phase 3 trial from Canada (NCT04362085) is recruiting patients to test the effect of therapeutic anticoagulant heparin in patients who are hospitalised with COVID-19. We await results of these trials with hope.EBioMedicine look forward to seeing translational and clinical studies that could accelerate the diagnosis, management and treatment of this devastating disease.Our understanding of COVID-19 has evolved rapidly over the past few months and new therapies have been proposed and tested. Nevertheless, many mysteries remain. Future scientific discoveries will shed new light on our understanding of COVID-19. In particular, we at EBioMedicine


The host innate immunity offers the first line of defense against infection. However, recent evidence shows that the host innate immunity is also critical in sensing the presence of cytoplasmic DNA derived from genomic instability events, such as DNA damage and defective cell cycle progression. This is achieved through the cyclic GMP-AMP synthase (cGAS)/Stimulator of interferon (IFN) genes (STING) pathway. Here we discuss recent insights into the regulation of this pathway in cancer immunosurveillance, and the downstream signaling cascades that coordinate immune cell recruitment to the tumor microenvironment to destroy transformed cells through cellular senescence or cell death programs. Its central role in immunosurveillance positions the cGAS-STING pathway as an attractive anti-cancer immunotherapeutic drug target for chemical agonists or vaccine adjuvants and suggests a key node to be targeted in a synthetic lethal approach. We also discuss adaptive mechanisms used by cancer cells to circumvent cGAS-STING signaling and present evidence linking chronic cGAS-STING activation to inflammation-induced carcinogenesis, cautioning against the use of activating the cGAS-STING pathway as an anti-tumor immunotherapy. A deeper mechanistic understanding of the cGAS-STING pathway will aid in the identification of potentially efficacious anti-cancer therapeutic targets. When functioning properly, the immune system protects the body against disease and pathogenic attack. The first line of defense is the innate immune system, which provides nonspecific defense mechanisms, including physical, chemical and immune responses, which protect the organism against microbial insult. Specialized RNA- and DNA-sensing receptors police subcellular compartments for infiltrating foreign nucleic acids, and, upon recognition, trigger immune signaling pathways that initiate a host defense. DNA infiltration into the cytosol can occur following a range of cellular changes or insults, including cellular senescence , 2, replThe innate immune system is triggered by the presence of nucleic acids in the cytoplasm through a range of pattern recognition receptors (PRRs) , includicGAS is a mediator of the DNA sensing pathway that is activated upon the presence of DNA in the cellular cytosol. cGAS is an enzyme of the nucleotidyltransferase family and senses the aberrant expression of viral and bacterial dsDNA within the cytosol in a sequence-independent manner. Binding of cGAS to dsDNA promotes cGAS oligomerization and activation, which catalyzes the formation of the cyclic dinucleotide (CDN), cyclic GMP-AMP  pA  p]; 2’3′-cGAMP), otherwise referred to as cGAMP –21. cGAS2+ and Mn2+, can stimulate cGAS activity , chronic cGAS-STING activation due to the persistence of cytosolic DNA causes the opposite effect, inflammation-mediated tumorigenesis. For example, inactivation of the BRCA2 gene causes impaired DNA repair and micronuclei accumulation, which activates the cGAS-STING pathway, resulting in cell cycle arrest, increased IFN signaling, and increased TNFα secretion, triggering cell death , 153. HoCompared with nascent or primary tumors, metastatic tumors are associated with chromosomal abnormalities such as aneuploidy and even polyploidy, which can arise from a mis-segregation of chromosomes during anaphase . Due to While cGAS-STING indisputably plays an important role in mediating the anti-tumor immunity, the above findings showing that chronic cGAS-STING activation and induction of the STING-dependent non-canonical NF-κB pathway seem to be pro-tumorigenic warrant further studies to clearly delineate the mechanisms driving these dichotomous outcomes.Based on the non-redundancy of STING in response to cytosolic DNA sensing, as evidenced by the lack of other cellular receptors for cGAMP , cancer Compared with normal gastric epithelia, STING expression is significantly reduced in surgically resected samples of gastric cancer . In gast+ T cell-mediated anti-tumor immunity and the establishment of an immunosuppressive environment with Treg and MDSC recruitment ] (ML-RR-CDA) has been shown to be a potential STING agonist in the clinic . Relativvenously .Because of the role of cGAMP export in triggering the immune response, a new study sought to exploit ENPP1, the enzyme responsible for extracellular degradation of cGAMP, by developing a small molecule inhibitor, STF-1623 . AdminisCDNs, such as cGAMP, carry negative charges and are thus hydrophilic, which restricts their diffusion into the cytosol of target cells. Thus, to enhance the cellular internalization of cGAMP, a cationic liposomal delivery vehicle was developed. This delivery vehicle not only improved tumor volume control but also survival in mice bearing PD-L1-insensitive tumors (including triple-negative breast cancer and B16F10 melanoma) as compared with soluble cGAMP or anti-PD-L1 therapy . Systemiin vitro, and improved CDN delivery and immune cell internalization [in vivo with a similar efficacy to a much higher dose (10×) of free CDN [Likewise, a biodegradable poly (beta-amino ester) nanoparticle-based CDN delivery system improved drug potency more than 100-fold as compared with free CDN administration lization . When cofree CDN .+ T cell response that was reactivated when the mice were re-exposed to A20 B-cell lymphoma cells [in vivo [+ T cells [Several studies have explored the utility of cGAMP/STING agonists as vaccine adjuvants. In a previous murine B cell lymphoma study, vaccination of mice with a STING agonist and anti-GITR yielded a CD8ma cells . Compare[in vivo . Another[in vivo . The adm T cells , 176. Si T cells , 176.A vaccine-based therapy involving the injection of irradiated B16 OVA cells containing a 90-bp 5′-modified AT-rich dsDNA—thus a cGAS ligand—into mice with metastatic B16 melanoma resulted in prolonged survival as compared with the control . CombineGenetic pathways often act collaboratively, and this holds true for mechanisms related to cancer cell survival, with communication between avoidance and resistance pathways likely to contribute to cancer cell growth and metastasis. This is where a synthetic lethality approach can be effective, even against hitherto untreatable cancers. Synthetic lethality describes the process by which two or more oncogenic pathways are simultaneously targeted to achieve or enhance a therapeutic response –180. GivThe primary aim of anti-cancer treatments—whether conventional chemotherapy, radiotherapy or immunotherapy—is to reduce tumor volume and release tumor-specific antigens to prime the immune system . Several+ T cells in tumor-draining lymph nodes (TDLNs) was also increased in a STING-dependent manner as compared with either therapy alone [via innate immune responses, lower doses of CDN, coupled with radiotherapy, could stimulate the adaptive immune response involving CD8+ T cells [A dose-dependent synergistic effect of CDN co-administration with radiotherapy was observed in mice bearing pancreatic adenocarcinoma or MC38 colon adenocarcinoma , 163. Thpy alone . Whereaset al. raises concerns about therapeutic approaches that use CDNs that only target STING and fail to discriminate between canonical and non-canonical downstream NF-κB pathways. The authors showed that the concomitant activation of the canonical and non-canonical NF-κB pathways suppresses IFNβ production, and dampens the therapeutic effect of radiotherapy [However, a study by Hou otherapy .Synergistic drug effects mean that the required dose of each constituent drug is reduced, and this helps to attenuate side effects or other toxicities associated with the administration of high doses . Becausevia non-homologous end joining and micro-homology end joining pathways, is another potential treatment target [via the death of proliferating immune cells [Finally, poly (ADP-ribose) polymerase (PARP), a key factor regulating the repair of DNA strand breaks t target , 180. PAt target , 180. Int target . Similart target . Howeverne cells , promptiNumerous factors within the TME affect T cell anti-tumoral responses. For instance, MDSCs express Fas ligands, which bind to Fas receptors on TILs to induce apoptosis , and thein vivo. Both ICB therapies achieved a reduction in tumor volumes by recruiting tumor-infiltrating CD8+ and CD4+ T cells [Consistent with the essential role of cGAS-STING signaling in innate immune sensing and priming T cell-mediated anti-tumor immunity, STING is needed to mediate the anti-tumor effects of ICB therapy. For example, STING-deficient mice treated with anti-CTLA4 and anti-PDL1 mAbs showed poorer B16 melanoma tumor control than WT mice . cGAS-ST T cells , 193.+ T cells, leading to improved tumor control. The ICB anti-tumor effect can be further enhanced in a dose-dependent manner by the intramuscular delivery of cGAMP at a distant site [Remarkably, combining ICB with CDNs can enhance the production of tumor antigen-specific CD8ant site . Similarant site . Apart fant site .et al. comprehensively studied the TME changes within TRAMP-C2 prostate tumors in response to c-di-GMP and anti-PD-1, anti-CTLA4 and anti-4-1BB combination therapy. TRAMP-C2 tumors predominantly contain myeloid cells (CD11b+F4/80+Gr-1- TAMs and CD11b+Gr-1+ MDSCs), with T cells  making up 5% of all CD45+ cells [+ T cells to Tregs, MDSCs, and M2 macrophages increased through Treg antibody-dependent cellular cytotoxicity, and resulted in M2 to M1 macrophage repolarization [+ T cells and clearance of tumor cells in immune-tolerant neu/N mice compared with ADU-S100 monotherapy, suggesting that ICBs can potentiate the anti-tumor effect of CDNs [Ager 5+ cells . In resprization . Similar of CDNs .https://clinicaltrials.gov/, NCT02675439 and NCT03172936).Overall, these studies support the synergistic actions of cGAS-STING activation and ICBs to attenuate immune tolerance in tumor cells and enhance the recruitment and priming of anti-tumor immune cells. Combination therapy consisting of cGAMP and ICBs thus represents a new paradigm in the management of tolerogenic cancers. In fact, the STING agonist ADU-S100 is currently being studied as monotherapy or in combination with anti-PD1  for the treatment of advanced, treatment-refractory metastatic solid tumors/lymphomas in two separate phase 1 clinical trials  and systemic lupus erythematosus (SLE), to organ-specific pathologies such as myocardial infarction (MI).Activating STING mutations have been implicated in an autoimmune condition known as STING-associated vasculopathy with onset in infancy (SAVI), in which the mutant STING localizes to the Golgi within the fibroblasts of affected patients, which in turn results in the constitutive activation of IFN expression even in the absence of cGAMP stimulation . Such auThe connection between the anti-tumor response and autoimmunity highlights the dual nature of the cGAS-STING-mediated response. On one hand, cGAS-STING activating therapies can trigger an intended pro-inflammatory response to cancers, but it can potentially also initiate a cascade of autoimmunity and inflammation, which can be as devastating as the cancers these treatments were designed to circumvent. As an example, TNFα, which is a recognized anti-tumor cytokine and a component of cGAS-mediated anti-tumor response, is also a central driver of RA pathogenesis. TNFα is normally expressed by DCs and T cells in response to cGAS-STING activation, but in the context of RA, it stimulates the expression of cGAS which proceeds to potentiate the inflammatory response in RA synoviocytes . WhetherWhile studies have not demonstrated a direct link between cGAS agonists and autoimmune conditions, these examples underscore the interconnected nature of the cGAS-mediated anti-tumor pathways and inflammatory diseases, with significant implications for applying cGAS/STING-based anti-cancer treatments: (1) more studies are needed to identify the full spectrum of human diseases mediated by cGAS-STING inflammation; (2) future studies should evaluate the long term effects of cGAS-STING agonists not only on cancer remission, but also on the development of other chronic conditions; and (3) strategies should be developed to adequately mitigate these potential risks associated with cGAS/STING-based therapies.in vivo, STING expression in cancer cells is not as critical for STING ligand-induced anti-tumor immunity as STING expression in the tumor stroma [Considering the differential expression of STING in various cell types, it is reasonable to question whether the use of STING ligands will be effective in the treatment of cancer. Fortunately, r stroma .Immunogenicity varies among different murine tumor models, and this in turn will lead to differing responses following STING ligation. For example, the murine MC38 colon adenocarcinoma, 4T1 metastatic breast cancer and CT26 colorectal carcinoma tumor models have higher proportions of tumor-infiltrating cells , and thuAdditionally, amidst the excitement surrounding the plethora of anti-cancer therapies capitalizing on stimulating the cGAS-STING pathway, it is critically important to consider the fine line separating anti-tumor immunity from autoimmunity and to thoroughly and cautiously evaluate all treatment options to better understand the risks of these treatment modalities.This review summarizes the critical role of the cGAS-STING pathway in mediating the autophagic, innate, and adaptive immune responses to cytosolic DNA arising from tumor cells. While there is great potential for using cGAS-STING agonists as anti-cancer agents, their use as clinical therapeutics should proceed cautiously due to the risk of tumorigenesis associated with chronic cGAS-STING activation and non-canonical downstream signaling. Defining the immune profile of tumors and elucidating the pathways controlling the anti- and pro-tumor effects of cGAS-STING activation will pave the way forward for its implementation in anti-cancer therapy.


Understanding the cellular and molecular mechanisms of tumor initiation and progression for each cancer type is central to making improvements in both prevention and therapy. Identifying the cancer cells of origin and the necessary and sufficient mechanisms of transformation and progression provide opportunities for improved specific clinical interventions. In the last few decades, advanced genetic manipulation techniques have facilitated rapid progress in defining the etiologies of cancers and their cells of origin. Recent studies driven by various groups have provided experimental evidence indicating the cellular origins for each type of skin and esophageal cancer and have identified underlying mechanisms that stem/progenitor cells use to initiate tumor development. Specifically, cyclooxygenase-2 (Cox-2) is associated with tumor initiation and progression in many cancer types. Recent studies provide data demonstrating the roles of Cox-2 in skin and esophageal malignancies, especially in squamous cell carcinomas (SCCs) occurring in both sites. Here, we review experimental evidence aiming to define the origins of skin and esophageal cancers and discuss how Cox-2 contributes to tumorigenesis and differentiation. Drugs that block a pro-inflammatory enzyme implicated in cancer initiation and progression could help suppress the development of skin and esophageal cancers that arise from abnormal squamous cells (skin cells and cells lining the respiratory and digestive tracts). In a review article, Hyeongsun Moon from the University of California, Davis, USA, and colleagues discuss experimental evidence from genetically engineered mouse models demonstrating that expression of an enzyme called cyclooxygenase-2 (Cox-2) is critical to the transformation of stem and progenitor cells in the skin and esophagus into cancer cells. Cox-2 is already the target of many drugs approved for treating inflammatory conditions such as arthritis. The preclinical data suggest that the same medications, or agents directed at mediators of Cox-2 signaling, may help tamp down the inflammation that can spur tumor-prone cells into turning malignant. Prevention of tumorigenesis represents an underappreciated opportunity to elicit a major impact in reducing cancer incidence and patient mortality. Moreover, inhibition of tumor initiation from cells that have already accumulated transforming mutations could provide a novel method for cancer prevention. To achieve this, it is important to fully understand and define the cells of origin for each cancer subtype and determine under which conditions a cell harboring transforming mutations is able to proliferate, invade surrounding tissues, and evade immune surveillance, leading to malignant cancer behavior.1. In the normal, disease-free state, these enzymes contribute to cell homeostasis; however, when homeostasis is perturbed by disease, they play critical roles in response but can also contribute to the development of a myriad of diseases, including cancer1. They provide a critical function as components in the enzymatic conversion of arachidonic acid to one of five unique prostanoid molecules. Two functionally redundant Cox enzymes exist, but each is unique in its spatial and temporal expression. Cox-1 is expressed as a housekeeping enzyme in most tissues during homeostasis, whereas Cox-2 is generally upregulated only in pathological conditions such as inflammation and cancer. Moreover, increased synthesis of prostanoids through Cox-2 activity can significantly contribute to the induction of inflammation and tumorigenesis1. Importantly, Cox-1/2 activity can be pharmacologically inhibited by nonsteroidal anti-inflammatory drugs (NSAIDS), such as naproxen or indomethacin, and Cox-2 activity can also be suppressed by selective inhibitors. The FDA-approved Cox-2 inhibitor celecoxib is used clinically to treat inflammatory diseases such as rheumatoid arthritis and osteoarthritis2. Although many studies have suggested the potential benefit of Cox-2 inhibition, especially in the prevention of colon cancer, whether suppression of this enzyme can be preventative or therapeutic for squamous cell carcinomas (SCCs) remains unclear. Here, we review the cellular origins of major primary cutaneous and esophageal cancers and discuss experimental evidence for two new roles of Cox-2 in the genesis of cutaneous and esophageal SCCs based on defined genetically engineered mouse models.Cyclooxygenase (Cox) enzymes are a class of molecules central to the environmental changes involved in tumor initiation3. Melanoma accounts for less than 5% of overall skin cancer cases, but the incidence of melanoma is increasing, with twice as many cases in the US this year compared to 30 years ago5. Additionally, despite the much lower incidence of melanoma than nonmelanoma skin cancers, melanoma accounts for the majority of skin cancer mortality5.The skin is the largest organ and is tasked with protecting our body. It is also the most common site of cancer diagnosis. Primary malignancies in the skin include basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma. Nonmelanoma skin cancers (keratinocyte carcinomas: BCCs and SCCs) are the most common, as over 3 million new cases are expected annually8. Notably, genetically engineered mouse models using conditional knock-in and knockout systems, which specifically target each cellular subset , can allow us to monitor the earliest stages of nonmelanoma and melanoma skin cancer development9.During the last decade, there has been great progress in understanding the cellular origins of various types of cancers, including both nonmelanoma and melanoma skin cancers. Particularly, genetic approaches with lineage tracing methods in mouse models have determined the earliest steps of tumorigenesis from stem/progenitor cells in multiple organ sites13. Constitutive activation of the G protein-coupled receptor Smoothened (SMO) through the Rosa26-SmoM2 allele appeared to be primarily involved in tumor formation from basal stem/progenitors within the interfollicular epidermis and infundibulum10. On the other hand, genetic inhibition of the tumor suppressor Patched 1 (PTCH1) using Ptch1+/− mice or expression of mutant GLI family zinc-finger 2  using Rosa26-LSL-rtTA; tetO-GLI2ΔN mice demonstrated a significant contribution of keratin 15 (KRT15), keratin 19 (KRT19) and leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive hair follicle stem cells in BCC development13. These studies reported that the constitutive activation of the Hedgehog pathway by oncogenic driver mutations (gain-of-function) or the absence of Hedgehog pathway suppressors could be involved in BCC formation from multiple cellular origins via resident stem/progenitor cells in both the hair follicular epithelium and interfollicular epidermis, especially in mechanosensory hot spots11.BCCs, the most common skin cancer, were postulated to originate from hair follicles due to the similarity in histologic characteristics of tumor tissues. However, oncogenic activation of the Hedgehog pathway revealed that both the interfollicular epidermis and hair follicular epithelium can form BCCs16. Tumorigenesis associated with the cutaneous application of 7,12-dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA), the most common chemical treatment used to induce SCC in a murine model system, is primarily caused by mutations in Hras15. Kras mutations are also induced by this chemical mutagen but at a significantly lower frequency15. In addition to DMBA-induced chemical mutations, various studies have documented tumorigenesis of SCC via genetic enhancement of the RAS pathway using the LSL-Kras-G12D allele  together with several different inducible Cre transgenic mouse models, including Krt5-tet-on, Krt14-CrePR, Krt14-CreER, Krt15-CrePR, Lgr5-CreER and Lgr6-CreER23. Oncogenic RAS expression through both DMBA chemical treatment and Kras gain-of-function can lead to the development of papillomatous tumors, which are considered a potential precursor lesion of SCCs. In addition, the expression of KrasG12D together with loss of function of the tumor suppressor p53 (oncogenic Ras/p53 combination) significantly accelerates tumor transformation from benign papillomatous tumors to invasive, spindle cell SCCs21. Intriguingly, upon oncogenic Ras/p53 expression, while Krt5-CreER and Krt14-CreER basal progenitors at the interfollicular epidermis primarily develop into papillomatous tumors, Lgr5-CreER, Krt15-CrePR, and Krt19-CreER hair follicle stem cells develop into invasive, mesenchymal-type SCCs23. Compared with Lgr5- and Krt15-positive hair follicle stem cells, Lgr6-CreER hair follicle stem cells located at the upper portion of hair follicles are less tumorigenic upon the same oncogenic Ras/p53 expression23. Hence, these studies suggest that multiple stem cells that differentiate into hair follicular epithelium and epidermal keratinocytes can contribute to SCC formation; however, each stem cell population located in different stem cell niches may have different tumorigenic potential and contribute to the diversity of SCC subtypes even when they harbor the same oncogenic combination 25. Constitutive activation of the mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways by oncogenic mutations in RAS and rapidly accelerated fibrosarcoma (RAF) genes  often causes oncogenic senescence in melanocytes. These benign nevi are known to require additional genetic changes, such as the loss of tumor suppressors, including cyclin-dependent kinase inhibitor 2A (CDKN2A) and phosphatase and tensin homolog (PTEN)25. The additional genetic alterations help benign melanocytic nevi cells overcome oncogenic senescence to become malignant melanocytic tumor cells. Cutaneous melanomas, however, are often diagnosed from patients who have no clinical history of benign moles or an identifiable precursor lesion27. These melanoma cells originating from clear skin are considered to originate from sustained unrecognized benign nevi or tumor-prone melanocyte stem cells. Recent studies driven by independent groups have experimentally demonstrated that melanoma can directly originate from melanocyte stem cells using Tyr-CreER and c-Kit-CreER promoters30. These independent studies30 used the same cell-type-specific expression of oncogenic profiles: a tumor driver mutation, BRAF , together with absence of the tumor suppressor PTEN . This combination is known to be able to induce invasive and metastatic melanoma cells31. Interestingly, melanoma cells directly originated from melanocyte stem cells in the absence of benign nevi. Furthermore, these melanoma-competent/susceptible melanocyte stem cells located within the hair follicles were able to translocate to the interfollicular epidermis, where they, in turn, formed cutaneous melanoma throughout the epidermis30. These new observations suggest the need to determine specific prevention strategies for melanomas arising from benign nevi and tumor-prone stem cell populations.While BCC and SCC originate from skin keratinocytes, cutaneous melanoma arises from melanocytes, the pigment-producing cells. Benign nevi may be precursor lesions that can progress to malignant melanocytic tumors when they gain additional mutations or genetic alterations35, which may have tumor promotive or inhibitory functions. Importantly, stem cell differentiation fate can be altered by various physiological and environmental stress factors that change the tumor microenvironment36. We have reported that stem cell quiescence can act as a tumor suppressor in skin SCC and melanoma formation28. While hair follicle and melanocyte stem cells can act as cancer cells of origin, when in a quiescent state, these stem cells resist tumor development28. These studies experimentally demonstrated the importance of extrinsic influences, since tissue injury and/or aberrant stem cell activation can significantly shorten the tumor latency period related to mutant stem cells28.Multiple resident stem cell populations within the skin layers can be involved in nonmelanoma and melanoma skin cancer development; however, each population located in distinct stem cell niches has different tumorigenic potential that can correlate with distinct tumor phenotypes. These distinctive tumorigenic capacities may be due to various reasons, including the differential intrinsic expression levels of certain molecules, innate lineage fate of each stem cell population, and the composition of the stem cell niche microenvironment 39. Numerous studies have determined the effects of UV radiation on skin cancer development. UV radiation can directly act as a mutagen by causing keratinocyte and melanocyte DNA damage39. However, UV exposure can also act on cancer development and progression indirectly through UV-induced inflammation, which may enhance aberrant activation of tumor-prone but quiescent stem cells harboring pre-existing oncogenic mutations. Experimentally, we have demonstrated that quiescent follicular melanocyte stem cells are less likely to develop cutaneous melanoma even when they express oncogenic mutations, BrafV600E together with Pten loss of function mutations29. However, during the period of melanocyte stem cell quiescence, cutaneous UV exposure induces aberrant activation and translocation of melanoma-prone stem cells into the interfollicular epidermis, which in turn causes significant malignant melanocytic tumor formation throughout the epidermis29. Transcriptomic comparison between quiescent melanocyte stem cells and early melanoma cells suggested significant involvement of inflammatory mediators during melanoma formation from tumor-prone stem cells28. It has also been reported that cutaneous SCC formation from hair follicle stem cells requires the activation of stem cells. While the oncogenic expression of KrasG12D along with p53 loss of function is sufficient to induce invasive SCC formation from hair follicle stem cells, stem cell quiescence significantly inhibits the initiation of SCC formation23. Intriguingly, the study found that suppressed tumor formation was dependent on the phosphoinositide 3-kinase–protein kinase B/Akt (PI3K/Akt) pathway, as Pten conditional knockout in tumor-prone, quiescent hair follicle stem cells directly induced SCC formation even during the stem cell quiescent period23. This suggests that UV radiation can also enhance the aberrant initiation of hair follicle stem cell-originating SCCs since UV radiation is known to activate the PI3K/Akt pathway40 and cutaneous SCC formation from murine hair follicles41.In both keratinocyte carcinomas and melanoma skin cancers, a major risk factor is ultraviolet (UV) exposure42. Previous reports have observed that Cox-2 is frequently overexpressed in both keratinocyte and melanoma skin cancers;45 thus, Cox-2 has long been considered a tumor promoter in skin cancers. It is well known that Cox-2 overexpression can increase tumor growth, decrease apoptosis, and advance progression through mechanisms including immune evasion and increased invasiveness through epithelial-mesenchymal transition (EMT)-like processes48. Experimentally, a rodent diet containing a selective Cox-2 inhibitor had suppressive effects on BCC formation in Ptch1+/− mice49. Similarly, treatment with an NSAID, naproxen, significantly suppressed UV-induced BCC and SCC formation50. The role of Cox-2 in skin tumor formation was also supported by transgenic Cox-2 overexpression51. Although Cox-2 overexpression alone had no significant association with spontaneous cutaneous SCC tumor formation in genetically engineered mice, transgenic overexpression of Cox-2 demonstrated a significant increase in sensitivity to DMBA/TPA-induced tumor formation in mouse skin51. As expected, a significant association of prostaglandin accumulation was observed in the epidermal layers during Cox-2-mediated accelerated tumor formation51. Prostaglandin E2 is known to bind and activate its G protein-coupled receptors, prostaglandin E2 receptors 1 to 4 . The tumor-promoting role of Cox-2 can partly work through each EP receptor, and inhibition of the receptor pathways has the potential to prevent cutaneous SCCs55. For example, Tober et al. reported suppressed UV-induced skin tumor formation by treatment with the specific EP1 antagonist ONO-871352. Sung et al. reported a significant reduction in DMBA/TPA-induced tumor formation in EP2-knockout but not EP3-knockout mice53, whereas transgenic overexpression of EP2 significantly increased cutaneous tumorigenic potential54. Similarly, transgenic overexpression of EP4 also significantly enhanced the SCC tumorigenic potential induced by the chemical carcinogen DMBA/TPA and UV radiation in mice55.Additionally, UV exposure also significantly induces Cox-2 expression and proinflammatory cytokines in the skin57. Genetic knockout of Cox-2 (prostaglandin-endoperoxide synthase 2 (Ptgs2) knockout) also significantly suppressed epidermal differentiation and DMBA/TPA-induced cutaneous papilloma formation59. More recently, Cox-2 knockout in Krt14-Cre+ epidermal basal cells induced a significant reduction in chemical carcinogen-induced tumorigenic potential59. Similarly, cell-type-specific Cox-2 knockout in Krt14-Cre+ epidermal basal cells significantly suppressed UV-mediated tumor formation in mouse skin60. Using a Cox-2 conditional knockout model (Ptgs2flox/flox) developed by the Herschman group61, we have also recently determined the role of cell-type-specific Cox-2 expression in Krt15-CrePR+ tumor-prone hair follicle stem cells expressing KrasG12D together with a p53 loss of function mutation62. Cox-2 is known to regulate the Akt-mTOR pathway, which is also required for efficient cutaneous wound healing64. Additionally, Cox-2 inhibition could have suppressive effects on the development of tumors from mutant hair follicle stem cells, as Akt pathway activation enhances SCC formation41. Intriguingly, genetic inhibition of Cox-2 suppressed tumor development and inhibited EMT-like properties during tumorigenesis62. Oncogenic Ras/p53 expression significantly and directly induces invasive, mesenchymal-like SCC formation from Krt15-positive stem cells, which tend to show loss of epithelial markers  but overexpression of mesenchymal markers 23. However, the same stem cell populations with a lack of Cox-2 expression tend to form less aggressive or typical epithelial-type SCCs (in contrast to spindle-type SCCs) that are typically well demarcated from the dermis. Together with previous studies60, this study62 demonstrates that Cox-2 expression acts as a tumor promoter that is both required for efficient tumor formation and involved in determining the subtype of cutaneous SCC tumors from the same cancer cells of origin  conditional loss of function, especially during superficial wound healing66. Cox-2 is known to be involved in the re-epithelialization of early phases of wound healing;67 hence, it is also be important to determine whether selective Cox-2 inhibition can regulate wound healing-mediated hair follicle stem cell migration and SCC formation.In cutaneous melanoma, we have also reported that dexamethasone treatment suppresses UV-induced melanoma formation from mutant, tumor-prone follicular melanocyte stem cells4. Although localized/early-stage tumors have a higher survival rate, the majority of patients present with advanced-stage/metastatic disease. Esophageal cancer prevention research has identified important behavioral risk factors, including smoking and drinking alcohol, and conditions that increase susceptibility, such as gastroesophageal reflux disease (GERD)70. Recent studies have also determined the cellular diversity of stem/progenitor cells in the esophagus  and their potential contribution to esophageal cancer formation75.Esophageal cancer is one of the most common lethal cancers worldwide, with an overall 5-year survival rate of up to 20%72. In genetically engineered mouse models, histopathological changes such as Barret’s metaplasia can arise from residual embryonic stem cells and/or a Krt7-positive subset of transitional basal stem/progenitor cells77. On the other hand, SCCs often arise from the squamous layer at the middle to upper  regions of the esophagus. Oncogenic mutations can induce hyperplastic to papillomatous tumors and SCCs from basal progenitors expressing Krt5, Krt15, p63, and SRY-box2 (Sox-2)75. In addition to anatomical differences between adenocarcinoma and SCCs . These two cancers are histologically and anatomically distinct. Although still controversial, the cellular origin of adenocarcinomas appears to be distinct stem cell populations located at the squamocolumnar junction80. GERD is a strong risk factor for Barrett’s adenocarcinoma;81 however, GERD is also associated with an increased risk of SCC74. These same risk factors are also associated with Cox-2 overexpression;82 thus, Cox-2 has long been considered a molecular target for esophageal cancer prevention85.In both esophageal SCCs and adenocarcinomas, the risk factors are related to individual behaviors and other conditions inducing constant tissue injury, which may act as a mutagen and increase tissue turnover rates. In SCCs, smoking cigarettes and drinking alcohol are the most well-known risk factors86. Similarly, a diet containing a selective Cox-2 inhibitor, L-748706 (L-706), suppressed NMBA-induced tumor development87. In addition, genetic knockdown of Cox-2 in human esophageal SCC cell lines suppressed tumor formation in vivo in xenograft mice88. Importantly, Liu et al. reported that Sox-2-positive basal progenitors contribute to murine foregut tumor formation, and furthermore, Sox-2, inflammation, and signal transducer and activator of transcription 3 (Stat3) cooperate to accelerate malignant transformation of basal progenitors73. Stat3 activation in acidic environment-induced inflammation correlates with the activation of Cox-2 and nuclear factor-κB (NF-κB)91. Cox-2 also facilitates esophageal SCC formation, as Cox-2 expression drives proliferation and reduces apoptosis in epidermal basal layers in skin tissues59. Notably, our recent study identified a novel role of cell-type-specific expression of Cox-2 in tumor-prone Krt5+/Krt15+ basal progenitors74 using a promoter transgene, Krt15-CrePR92, and a Cox-2 conditional knockout allele, Ptgs2flox/flox61. Upon expression of the oncogenic Ras/p53 combination (KrasG12D expression with a lack of p53 expression), some basal progenitors were able to develop tumors, and tumorigenesis was significantly accelerated by microenvironmental acid-induced stress74. However, cell-type-specific knockout of Cox-2 suppresses oncogenic Ras/p53-mediated tumor formation in a genetically engineered mouse model and in 3D organoids74. Intriguingly, without significant tumor formation, tumor-prone basal progenitors frequently undergo squamous differentiation and present high expression of the differentiation marker loricrin (primarily expressed in fully differentiated squamous epithelium)74. This study demonstrates the importance of inflammation-mediated intrinsic expression of Cox-2 in tumor-prone basal progenitors during esophageal SCC formation. Since Cox-2 can be upregulated in response to various extrinsic and intrinsic stress factors, it is important to note that not only mutations but also inflammatory conditions associated with these stress factors  can further accelerate tumor formation from mutant stem/progenitor cells in a Cox-2-dependent manner. Therefore, controlling Cox-2 expression could be beneficial for preventing esophageal SCC formation in patients who may be prone to constant esophageal tissue injury.Experimental studies consistently demonstrate that Cox-2 inhibition has the potential to suppress esophageal SCC formation. In previous studies, the role of Cox-2 in esophageal SCC was tested in an N-nitrosomethylbenzylamine (NMBA)-induced rat SCC model. The oral administration of a selective Cox-2 inhibitor, 4-[4-cyclohexyl-2-methyloxazol-5-yl]-2-fluorobenzenesulfonamide (JTE-522), could inhibit NMBA-induced tumor formation in the rat esophagus89. Cox-2 can also be significantly involved in Barrett’s metaplasia and esophageal adenocarcinoma. While a Cox-2 conditional knockout model has not yet been utilized in genetically engineered murine models of Barrett’s metaplasia , the role of Cox-2 has been examined in biopsy samples and surgically induced Barrett’s metaplasia rat models. Since bile acid can induce esophagitis in a short timeframe and metaplastic changes in the long term, surgical techniques such as esophagojejunostomy are often used to generate models in both mice and rats. The aim of the surgery is to enhance the exposure of the esophageal mucosa to bile acids. In the surgery-induced model, pharmacological inhibition of Cox-2 has shown potential to inhibit the carcinogenic effects of GERD94. These studies suggest that Cox-2 expression is upregulated in various physiological conditions and involved in pathological changes in both esophageal SCCs and adenocarcinomas.Similarly, constant low pH-induced stress from exposure to gastric and bile acids in GERD patients can induce overexpression of Cox-2 in the esophageal epithelium62 and esophageal SCCs74 have demonstrated that Cox-2 inhibition promotes a more well- or moderately differentiated phenotype than high levels of Cox-2. A lack or low levels of differentiation are hallmarks of cancers with poorly differentiated phenotypes and poor prognosis. Treatments aimed at stimulating tumor differentiation through the use of agents such as all-trans retinoic acid may be effective in cancer management95. It is also possible that all-trans retinoic acid and Cox-2 may have opposite roles or act synergistically in stem cell differentiation, although this has not been documented in cutaneous and esophageal stem/progenitor cells96. Therefore, it is important to clearly understand the details of Cox-2 mechanisms in tumorigenesis. Furthermore, whether Cox-2 inhibitors are effective in advanced-stage SCCs by driving differentiation warrants further investigation. Additionally, understanding the role of Cox-2 in cancer stem cells or tumor-propagating cells may also provide useful information for therapeutic targeting. Thus, future studies should define whether Cox-2 can regulate stemness and the differentiation of cancer cells. The effects of targeting Cox-2 may reverse disease status by suppressing stem-like features and enhancing the differentiation status of advanced cancer cells.Here, we discussed recent studies that explored the cellular origin of cutaneous and esophageal cancers and identified roles for cell-type-specific Cox-2 expression during stem/progenitor cell-originating cancer development. Particularly, these studies demonstrate that Cox-2 can promote the initiation of aggressive tumor formation from tumor-prone stem/progenitor cells in murine skin and enhance the formation of esophageal SCC near the squamocolumnar junction Figs.  and 2. T97, and Cox-2 is an important mediator of inflammation and cancer. We and others also reported that cell-type-specific Cox-2 knockout in stem/progenitor cells may suppress tumor-promoting inflammation in the niche of cancer cells of origin74. Suppressed tumor formation by Cox-2 inhibition can be, in part, indirect through a suppressive effect on tumor-promoting inflammation given the crosstalk between immune cells and stem cells. However, the effect of Cox-2 expression on stem cells in their niche needs to be determined in various tumor-initiation sites. While Cox-2 may be a promising molecular target to prevent stem/progenitor cell-originating tumor formation, it is important to clarify whether and how Cox-2 expression in stem cells is required for their normal function and how they regulate the stem cell niche. Importantly, cancer incidence in many organ sites increases with age. This phenomenon may prove to be associated with stem cell aging and Cox-2 expression99. Therefore, careful measurement of the role of Cox-2 in stem cells and their microenvironmental niche is essential to achieve better preventative methods for skin and esophageal malignancies in aging populations.We now have a better understanding of how stem/progenitor cells contribute to tumor formation and how inflammation may accelerate tumorigenesis from these tumor-competent stem/progenitor cells. Inflammation is one hallmark of cancerFurthermore, understanding the role of downstream molecules of Cox-2 in stem/progenitor cells and stem/progenitor cell-originating cancers may provide better preventative therapeutic targets. While selective Cox-2 inhibitors exist and are FDA approved for some conditions, NSAIDs and Cox-2 inhibitors still have common side effects limiting their long-term use, which is likely required for prevention. Therefore, future studies will need to contribute to a better understanding of Cox-2 pathways, including defining the role of cell-type-specific EP receptors. While this review does not focus on EP receptors in cancers, Cox-2 also acts through EP receptors. Future work should also seek to determine whether selective targeting of EP receptors can provide clinical benefit in stem cell-originating cutaneous and esophageal malignancies in both SCC and non-SCC tumors. Overall, together with identifying the clinical benefit of Cox-2 suppression in skin and esophageal malignancies, future studies should identify what downstream molecules need to be targeted to efficiently maintain normal stem cell functions but significantly inhibit tumor formation from tumor-prone stem cells.


Epidemiological results revealed that there is an inverse correlation between high-density lipoprotein (HDL) cholesterol levels and risks of atherosclerotic cardiovascular disease (ASCVD). Mounting evidence supports that HDLs are atheroprotective, therefore, many therapeutic approaches have been developed to increase HDL cholesterol (HDL-C) levels. Nevertheless, HDL-raising therapies, such as cholesteryl ester transfer protein (CETP) inhibitors, failed to ameliorate cardiovascular outcomes in clinical trials, thereby casting doubt on the treatment of cardiovascular disease (CVD) by increasing HDL-C levels. Therefore, HDL-targeted interventional studies were shifted to increasing the number of HDL particles capable of promoting ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux. One such approach was the development of reconstituted HDL (rHDL) particles that promote ABCA1-mediated cholesterol efflux from lipid-enriched macrophages. Here, we explore the manipulation of rHDL nanoparticles as a strategy for the treatment of CVD. In addition, we discuss technological capabilities and the challenge of relating preclinical in vivo mice research to clinical studies. Finally, by drawing lessons from developing rHDL nanoparticles, we also incorporate the viabilities and advantages of the development of a molecular imaging probe with HDL nanoparticles when applied to ASCVD, as well as gaps in technology and knowledge required for putting the HDL-targeted therapeutics into full gear. Epidemiological studies identified several independent risk factors for cardiovascular disease (CVD), including hypertension, age, smoking, insulin resistance, elevated low-density lipoprotein cholesterol (LDL-C) levels, and triglyceride levels . The majAdditional evidence for the atheroprotective effects of apoAI or HDL derives from mouse models of experimental atherosclerosis and clinical trials. One of the most effective approaches is to promote new HDL particle formation by enhancing endogenous production of apoAI, which is supported by using human apoAI transgenic mice studies as well as virus-mediated overexpression of apoAI in a mouse model of experimental atherosclerosis ,14,15. IHDL protective functions, including anti-inflammatory and mediation of reverse cholesterol transport (RCT), likely contribute to the regression of established plaques and are central in protecting against CVD ,21. For Much progress has been made to increase HDL-C levels in plasma using orally-administered small molecules such as niacin and cholesteryl ester transfer protein (CETP) inhibitors ,34. LargRecent failures of CETP inhibition aimed at increasing HDL-C levels to decrease the risk of CVD have shifted the focus of HDL research towards a better understanding of mechanisms to preserve HDL function(s), rather than raising HDL-C levels. HDLs mediate the transport of cholesterol from peripheral tissues to the liver from which it can be secreted as bile acid, the process of which is known as reverse cholesterol transport (RCT). There are basically three steps in this process: (1) cholesterol efflux, where apoAI and HDLs interact with ABCA1 and ABCG1 to remove excess cholesterol from lipid-enriched macrophages as shown in −/− mice [−/− mice. Rev-D4F also prevented LDL oxidation, reduced the expressions of endothelial cell vascular cell adhesion molecule 1 (VCAM-1) and monocyte chemotactic factor 1 (MCP-1), and improved vascular inflammation in Apoe−/− mice [Recent studies suggest that apoAI mimetic peptides serve as a promising new therapeutic approach by promoting the formation of pre-β HDLs, increasing cholesterol efflux, and preventing lipoprotein oxidation . The seq−/− mice . In addi−/− mice . More im−/− mice . Taken tEscherichia coli, and synthesized apoAI peptides. Development of rHDL nanoparticles that mimic pre-β HDLs, could promote cholesterol efflux from lipid-enriched macrophages, thereby reducing atherosclerosis. Many different rHDL nanoparticles  have been constructed and investigated in animal studies and human clinical trials [Proteomic studies found HDLs contain hundreds of proteins and lipids . Therefol trials .Emerging therapeutic approaches targeting rHDL have been investigated to reduce the risk of cardiovascular events in the past decades ,53. One To overcome the adverse effects of ETC-216, another recombinant apoA-IM, MDCO-216, was developed and its efficacy was examined in patients with coronary artery disease ,59. MDCO−/− mice. More importantly, there was a significant reduction in macrophage content and a reduction in VCAM-1 expression in the plaque after treatment of mice with CER-001 [CER-001 was made by recombinant human apoAI, sphingomyelin (SPM), and dipalmitoylphosphatidyl glycerol (DPPG) (protein to phospholipid = 1:2.7). CER-001 increased the reverse lipid transport and promoted atherosclerosis regression in Ldlr CER-001 . However CER-001 . Similar CER-001 .CSL-111, a reconstituted HDL particle, is combined by native apoAI and phospholipids. Recent studies demonstrate that rHDL CSL-111 infusion reduced the total number of circulating leucocytes and neutrophils in mice following ischemic reperfusion (I/R) ,67. CSL-A new formulation of human rHDL CSL-112 was developed by reconstituting human plasma-derived apoAI with phosphatidylcholine to form disc-shaped HDL particles. There are two molecules of human apoAI, about 110 molecules of phosphatidylcholine and sucrose as a stabilizing agent. CSL-112 significantly increased the cholesterol efflux from J774 macrophages . InfusioThese major rHDL-based therapies  are remarkably different in composition, dosing, pharmacokinetics, and pharmacodynamics as shown in −/− mice [−/− mice [The application of rHDL nanoparticles for delivering therapeutic compounds for the treatment of cancer has been studied extensively ,77,78. R−/− mice . Importa−/− mice .−/− mice with advanced atherosclerotic plaques [Statins have potent anti-inflammatory functions, but these cannot be fully exploited with oral statin therapy owing to a low systemic bioavailability. Interestingly, an injectable rHDL nanoparticle was synthesized to deliver simvastatin, and the effect of simvastatin-rHDL on atherosclerotic plaques was examined in mice. This study demonstrates that statin-loaded reconstituted HDL nanoparticles improved inflammation in atherosclerotic plaque . More in plaques . rHDL na plaques .Arachidonic acid (AA) was engineered into the rHDL complex to increase the efficacy of statins. AA-LT-rHDL (arachidonic acid-lovastatin-rHDL) exhibited lower reactivity with LCAT and more potent inhibition effects on foam cell formation in the presence of LCAT because of less undesired LT leakage during the remodeling of rHDLs induced by LCAT and more cellular drug uptake . In addiHDLs are highly heterogeneous and transport a large variety of lipids, proteins, and microRNAs . Anti-seMounting evidence shows that early stages of the lesion development is dominant by monocyte recruitment followed by monocyte differentiation into macrophages in mice, whereas macrophage proliferation is more predominant in advanced atherosclerotic plaques ,93,94. MLXRs, oxysterol-activated nuclear receptors, play an important role in RCT through promoting ABCA1 and/or ABCG1-mediated cholesterol efflux. In vivo PET imaging probes radiolabeled with zirconium-89 (89Zr) on discoidal HDL nanoparticles were made by the reconstituting apoAI and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine, the chelator deferoxamine B, and 89Zr . It was HDL-targeted drug CETP inhibitors except anacetrapib did not decrease cardiovascular events in clinical trials. Convincing results demonstrate that increased HDL cholesterol levels do not always correlate with enhanced protective HDL properties ,40,41, tAlthough several rHDL nanoparticles failed to regress the atherosclerotic plaques in humans, it should be noted that these clinical trials are relatively short-term studies; the duration of these trials was only 4–6 weeks. There is solid evidence that HDL beneficial effects have to do more with the achievement of a continuous flux and steady export of cholesterol, rather than absolute levels of HDL cholesterol . Whether


Self-reported memory problems and depressive symptoms tend to co-occur in older adults; however, this relationship may depend on personality traits and the type of self-reported memory assessed. Using a coordinated analytic framework, this study examined whether neuroticism, extraversion, and conscientiousness moderated the associations of older adults’ self-reported memory with depressive symptoms at between- and within-person levels across three large, longitudinal datasets (range=8-12 years of follow-up) of community-dwelling older adults with no evidence of cognitive impairment . Assessments of depressive symptoms (GDS-15 or CES-D) and self-reported memory  were taken annually or biennially; personality was assessed via the IPIP or NEO Five-Factor Inventory. Results were largely consistent across datasets. Specifically, between persons, self-reported memory problems  were related to higher depressive symptoms only among older adults higher in neuroticism. In one dataset, results supported a protective effect of conscientiousness such that higher frequency of forgetting was related to lower depressive symptoms among older adults higher in this trait. Within persons and across datasets, at times when perceived memory decline was reported, or current memory rating was lower, depressive symptoms tended to be higher only in older adults higher in neuroticism. Results demonstrate the importance of considering personality traits and the type of self-reported memory when examining associations among reports of memory problems and depressive symptoms in cognitively intact older adults.


Current cancer therapeutics often insufficiently eradicate malignant cells due to the surrounding dense tumor stroma. This multi-componential tissue consists of mainly cancer-associated fibroblasts, the (compact) extracellular matrix, tumor vasculature, and tumor-associated macrophages, which all exert crucial roles in maintaining a pro-tumoral niche. Their continuous complex interactions with tumor cells promote tumor progression and metastasis, emphasizing the challenges in tumor therapy development. Over the last decade, advances in oncolytic virotherapy have shown that oncolytic viruses (OVs) are a promising multi-faceted therapeutic platform for simultaneous tumor and stroma targeting. In addition to promoting tumor cell oncolysis and systemic anti-tumor immunity, accumulating data suggest that OVs can also directly target stromal components, facilitating OV replication and spread, as well as promoting anti-tumor activity. This review provides a comprehensive overview of the interactions between native and genetically modified OVs and the different targetable tumor stromal components, and outlines strategies to improve stroma targeting by OVs. Despite decades of research into cancer, and the development of a tremendous amount of treatment options, cancer remains a leading cause of death, with approximately 9.6 million deaths worldwide in 2018 ,4. HowevA major determinant of therapeutic resistance is the dense stromal tissue surrounding and imbedding the malignant cancer cells. Solid tumors are composed of tumor cells and up to 90% of non-epithelial cells that collectively form the tumor stroma . The tumOncolytic virotherapy (OV therapy) is a novel and promising therapeutic approach that has recently gained interest for its multi-faceted mechanism of action that induces immunogenic cell death and thereby strong anti-cancer immune responses. Oncolytic viruses (OVs) selectively target, replicate in and kill tumor cells while sparing normal tissues. In fact, OVs benefit from the tumor’s altered microenvironment, which displays certain tumor-specific receptors and modified cellular pathways ,11. OVs In this literature review, we will discuss the OVs that specifically target tumor stroma components and outline possibilities to improve this targeting, which is ultimately expected to result in enhanced anti-cancer activity.OVs are a collection of ‘cancer-killing’ viruses, known for their multi-faceted attack on tumors and their components. Through direct cancer cell lysis, they can boost a more effective anti-tumor immune response ,16. OVs A large variety of oncolytic viral families have been investigated in various preclinical and clinical cancer studies . These cSolid tumors secrete cytokines to suppress the anti-tumor functions of immune cells and recruit stromal cells, forming a desmoplastic physical stromal barrier . This muFibroblasts are the major multi-functional component of tumor stroma. It has been shown that local fibroblasts can develop into CAFs, although additional CAF progenitors have been identified as well, such as mesenchymal stem cells, adipocytes, epithelial and endothelial cells . CAFs stIn recent years, interest has increased in the heterogeneity of the CAF cell population. The numerous sources of origin and the tumor-distinctive activation processes result in a large variety of phenotypic markers expressed by the CAFs ,38,43. SFive different CAF subsets have been distinguished based on the origin of the precursor fibroblasts, activation stages, surface markers and secretory phenotype. An overview of the characteristics and functions of these CAF subsets is provided in The ECM comprises up to 60% of the solid tumor mass and is generated by CAFs and, to a lesser extent, tumor cells . The denVascular endothelial cells (VECs) can be transformed into more rapidly proliferating and migrating tumor endothelial cells (TECs) upon specific targeting and stimulation of signaling pathways by tumor cells and surrounding activated stromal cells . TogetheTumors steer both the innate and adaptive immune cell composition of the TME. In solid tumors, half of the tumor mass is represented by types of macrophages known as TAMs, which polarize into the pro-tumoral and anti-inflammatory M2 phenotype. The described recruitment and polarization are promoted by the tumor and immune cell secreted cytokines, growth factors and metabolites such as VEGF, colony stimulating factor-1 (CSF-1) and chemokine CCL2 . TAMs caSeveral studies have demonstrated the inherent capability of OVs to target the tumor stroma. Below, an overview is provided on the different stromal components for which natural targeting by OVs has been described.CAFs distinguish themselves from normal cells through their cellular origins, phenotypic plasticity, and diverse functions, as previously discussed. To date, natural CAF tropism has been identified for only one OV: VSV. VSV has been shown to successfully infect CAFs in patient-derived xenograft models. VSV replication in pancreatic tumor cells and stroma is enhanced by the cross-talk between tumor cells and CAFs . This crFour OVs with natural VEC specificity or binding abilities have been described, including reovirus, VSV, HSV, and vaccinia virus (VV). Wild-type reovirus has been found to effectively infect and lyse vascular endothelial cells (VECs) upon artificial elevation of VEGF levels. Subsequently, reoviral delivery and tumor lysis was enhanced in vitro and in murine syngeneic melanoma tumor models . SimilarThe 3D imaging of a murine colon cancer model has revealed direct VSV infection of VECs in tumor vasculature, but not normal vasculature. VSV infection induced coagulation in the neovasculature, reducing tumor perfusion and proliferation of malignant cells within the tumor core . The mecIn addition to VSV, VV has been studied as a potential treatment for colon cancer. Bell and colleagues have demonstrated VEGF-induced VV internalization in VECs of mice bearing colon carcinoma tumors. High levels of VEGF were shown to signal through VEGFR2, followed by ERK1/2 and STAT3 signaling, and upregulation of the transcription repressor PRD1/BF1. Thereby, the IFN-mediated anti-viral response of VECs was suppressed, resulting in enhanced sensitivity to VV infection .In addition to these observations of direct virus infection in VECs, OVs have also shown anti-angiogenic effects. Contrast-enhanced ultrasound performed by Thorne and colleagues showed an unexpected rapid vascular collapse and prevention of revascularization upon VV infection in murine breast and renal tumor models. This effect was associated with a VV-induced reduction in VEGF levels during the entire period of viral infection . HoweverStudies focusing on the effect of oncolytic HSV-1 on tumor angiogenesis show contradictory results. While one study has shown a direct anti-angiogenic effect of oncolytic HSV-1 on both murine and human VECs in vitro and in vivo , other rDespite the natural stroma targeting of the above discussed OVs, the dense tumor stroma can limit the viral spread, replication and infectivity . Stroma CAFs represent a promising target for OV therapy as they are the major stromal component. The studies discussed below show how OVs can target CAFs to enhance their replication, or to diminish the pro-tumoral effects and activities of CAFs. To date, they have not examined the targeting of specific CAF subsets. As previously discussed, each CAF subset exerts specific functions. Future studies are warranted to examine how this affects the therapeutic effect of OV-mediated targeting.A few studies have described bioselection as a powerful tool to select and develop oncolytic adenoviruses with improved stroma-targeting capacities . By repeFurthermore, combination treatment with the chemotherapeutic agent gemcitabine in Syrian hamsters bearing syngeneic pancreatic tumors that harbored CAFs was shown to enhance the therapeutic efficacy of ICOVIR-15i. This effect presumably occurs through enhanced tumor cell killing, which may lead to a reduced intratumoral interstitial pressure, followed by enhanced adenoviral penetration. In line with this, ICOVIR-15i-mediated targeting of both CAFs and tumor cells could result in less-dense tumor and stromal tissue, enhancing both OV and gemcitabine penetration . CAF-medWhile bioselection can be a powerful tool for modifying potential OVs, genetic engineering methods have yielded far more of these stroma-targeted OVs. Studies completed by Lopez and colleagues demonstrate the importance of CAF targeting in the oncolytic efficacy of replicative genetically modified adenoviruses. They generated Ad-F512, which expresses a secreted protein acidic and rich in cysteines (SPARC) promoter to selectively replicate in SPARC-expressing cells. SPARC is mainly expressed by stromal cells, including CAFs, and to some extent tumor cells . Ad-F512In another approach, the adenovirus strain AdTATMMP has been engineered to selectively enter cells upon proteolytic cleavage of its fiber by MMP2 and MMP9. Since MMP2 and MMP9 are involved in the continued growth of the tumor and metastasis, coupled with the fact that high expression of both is related to poor patient survival in various types of cancers, an OV that targets these markers could be of high clinical value . In compAnother way to target CAFs has been exploited with an adenovirus expressing a bispecific T cell engager (BiTe) targeted to FAP. FAP, as previously discussed, is one of the general markers expressed by CAFs. This virus, EnAd-CMV-FAP-BiTE, was shown to trigger T cell-mediated killing of CAFs in human malignant ascites as well as solid prostate cancer tissue . A similCAFs have also been targeted using a genetically modified measles virus (MV) that is retargeted against the murine or human uPAR  . The MV-In a recent follow-up study, MV-CD46-muPA has been developed, which was shown to target both human CD46-positive colon tumor cells and murine uPAR-expressing CAFs in vitro and in vivo. Systemic administration of MV-CD46-muPA to mice bearing human colon tumors improved tumor cell killing and overall survival compared to MV-GFP, indicating the importance of killing both tumor and stromal cells. Notably, the effects of MV-CD46-muPA were not compared with those of MV-m-uPA, MV-h-uPA, or combined MV-m-uPA/MV-h-uPA treatment . AnotherAnother way to target CAFs has been shown using genetically modified recombinant vaccinia virus, known as GLV-1h282, that expresses FAP-specific single-chain antibody (scAB). Upon intravenous injection of mice bearing human lung epithelial tumor xenografts, GLV-1h282 has demonstrated enhanced tumor regression compared to the unmodified GLV-1h68. This regression was likely related to OV targeting of FAP-expressing CAFs present in the model, as the tumor cells were shown to be FAP negative. Interestingly, the greatest tumor regression was shown after treatment with the engineered vaccinia viruses encoding scABs targeting both VEGF and EGFR (GLV-1h444), or VEGF and FAP (GLV-1h446). Although no direct CAF targeting was described, the observed tumor growth inhibition using GLV-1h446 was presumed to be partially attributed to CAF targeting .Altogether, these studies highlight the potential of generating OVs with enhanced stroma-targeting capacities through bioselection and genetic engineering. Importantly, the composition of CAF subsets and functions differ between tumors, as mentioned before. A description of the specific human CAF subsets involved in the experiments is lacking, rendering it challenging to extrapolate the findings to other tumors.It has become clear that genetically engineered OVs are able to target CAFs, which can improve OV distribution, infectivity in the tumor stroma, and therapeutic outcome in a variety of tumor types. However, the performed studies lack a clear CAF subset description, as for example FAP is a general CAF marker for both anti-tumoral myCAFs and pro-tumoral iCAFs . MoreoveThe recently identified pro-tumoral iCAFs represent a favorable target for OV therapy. In order to specifically target iCAFs, OVs should be directed against one of the specific iCAF markers . AGTR1 iThe pro-tumoral CD10+GPR77+ CAFs pose another interesting CAF subset for future OV therapy. GPR77, a surface marker and an essential signaling molecule for this specific CAF subset  50], co, co50], OV-expressed factors such as proteases can be used to degrade ECM components. For example, MMP9 has been shown to facilitate ECM degradation, and to enhance HSV delivery, distribution, and oncolytic effect in mice bearing human brain tumors . RecentlConversely, MMP suppression has been exploited to counteract the pro-tumorigenic roles of MMPs. Tissue inhibitor metalloproteinases 1 to 4 (TIMPs1-4) tightly regulate the proteolytic activity of MMPs in healthy human tissues. A decreased expression of TIMPs, and therefore over-active MMPs, has been shown to result in invasive, metastatic, and poor prognostic tumors . TherefoAltogether, targeting the ECM requires caution. The efficacy of either MMP expression or suppression to promote OV spread in the tumor stroma may depend on the tumor-specific ECM composition, requires adequate dosing and timing, and should be weighed against the potential release of tumor-promoting factors . MMP expMultiple approaches exist to target the tumor vasculature using genetically engineered OVs . These aFirstly, as previously discussed for CAFs, a variant of the SPARC promotor expressing Ad-F512, Ad(I)-F512-TK, is shown to effectively infect and kill both CAFs and endothelial cells in vitro. Combination treatment of this OV with gemcitabine of mice bearing a mix of pancreatic tumor cells and endothelial cells showed induced complete tumor remission compared to mice bearing tumor cells only . This imAnother OV which has also been shown to target VECs, in addition to CAFs, is the earlier discussed MV-uPA. Compared to MV-GFP, MV-m-uPA enhances endothelial cell infection both in vitro and in systemically infected mice bearing human breast tumors. Moreover, MV-m-uPA treatment resulted in superior anti-tumor effect and prolongation of survival compared to mock infection. No toxicity was observed, indicating specific targeting of uPAR-expressing endothelial cells . AltogetThe oncolytic bG47Delta-dnFGFR, an HSV expressing a FGFR therapeutic transgene, is shown to be another approach to inhibit the tumor vasculature. Upon treatment with this OV, mice bearing human glioblastoma tumors showed both angiogenesis and tumor growth inhibition. These results could be related to inhibition of FGF signaling in both VECs and tumor cells . ImportaAnother way to target VECs, currently studied in clinical trials, is the multi-functional engineered JX-594, an oncolytic VV with a disrupted viral thymidine kinase (TK) gene . JX-594 OVs have also shown anti-vascular effects by decreasing pro-angiogenic cytokines or proteins in tumor tissues. One approach is to target the highly expressed MMP14 in human glioma cells using an oncolytic adenovirus encoding an MMP14-silencing small hairpin RNA (shRNA), CRAd-S-5/3shMMP14. MMP14 expression is known for its angiogenesis-promoting effect, therefore silencing of this protein is shown to reduce pro-angiogenic IL-8 and VEGF levels in infected glioma cells in vitro, as well as angiogenesis and glioma proliferation in human glioma xenografts .Another adenovirus has been engineered to carry the anti-sense sequence for uPAR and MMP9, Ad-uPAR-MMP9. MMP9 is a collagen type IV-degrading enzyme, by which it contributes to metastasis, as well as an effective diagnostic biomarker for non-small-cell lung cancer (NSCLC) . Ad-uPARAn additional set of approaches involve engineered OVs targeting pro-angiogenic cytokines in order to target the tumor vasculature. Firstly, a study of Hemminki and colleagues has shown a clear anti-angiogenic effect using vvdd-VEGFR-1-Ig, an engineered VV targeted against kidney tumor cells that is armed with VEGFR-1-Ig. This VEGFR-1 is known to scavenge the highly expressed VEGF molecules, inhibiting VEGF-induced angiogenesis. Upon medium or low doses of systemic OV administration in renal tumors of immunodeficient mice, tumor regression, anti-tumor cytokine responses and anti-angiogenic effects were detected .In line with this, infection with the earlier introduced VVs encoding antibodies that target VEGF and EGFR (GLV-1h44), or VEGF and FAP (GLV-1h446) diminished angiogenesis and tumor cell proliferation in human prostate tumor xenografts. GLV-1h446, expressing anti-VEGF and anti-FAP, was specifically shown to decrease tumor proliferation in both infected and uninfected distal tumor areas, demonstrating the enhanced systemic therapeutic effects of this VV . InteresIL-12 has been demonstrated to enhance Th1-mediated anti-tumor responses and to inhibit tumor angiogenesis in severe-combined immunodeficient mice . This haFinally, the tumor vasculature has also been targeted by OVV-CXCR4-A-mFc, a VV expressing a CXCR4 antagonist, which is directed against the VEGF-induced pro-angiogenic CXCL12/CXCR4 signaling pathway in human breast tumor cells. Upon intravenous administration, this virus blocked CXCL12/CXCR4 signaling, inhibiting the recruitment of circulating endothelial progenitors (CEPs) into the stroma and destructing the tumor vasculature in human breast tumor bearing mice .Targeting of the highly abundant TAM population and their interactions with the tumor cells and other stromal components may help to reduce primary tumor growth, metastases, and recurrence. Multiple therapeutic approaches to reduce the pro-tumoral effects of TAMs have been reviewed by Portella and colleagues, including TAM depletion, reduction in macrophage recruitment, or modulation of the pro-tumoral M1 to M2 macrophages polarization . The oncDespite the importance of TAMs in steering the pro-tumoral and immunosuppressive environment of stromal tissues, no (engineered) OVs have been documented yet in which TAMs are directly targeted by OVs. Taken together, future studies focusing on OVs targeting TAMs by, for example, expressing transgenes directed against the previously discussed TAM markers such as CD163, CD204 or CD200R could be of great value.OVs represent a promising multi-faceted class of tumor therapeutics to overcome the dense tumor stroma . ExcludiBy targeting the rich ECM and its components such as MMPs, the density and stiffness of the tumor ECM will presumably decrease, easing OV, anti-tumor drug and lymphocyte migration towards the tumor . The subIn particular, OV-mediated targeting of CAFs appears promising, as CAFs are known as the ‘small factories’ of the dense ECM. In addition, CAFs have been identified as the major players in maintaining a pro-tumoral niche as well as regulating the complex tumor–stroma interactions. Multiple non-specific CAF markers have been identified. However, the heterogeneity of CAF subsets and their tumor-distinctive functions in different tumor types highlight the importance of examining and targeting more specific CAF populations that may differ per tumor tissue. The best-studied CAF subsets are the myCAFs and iCAFs present in pancreatic tumors. Future OVs should preferably be engineered to target the pro-tumorigenic iCAFs or CD10+GPR77+ CAFs by targeting specific surface markers, or the signaling pathways involved in their formation and function.Importantly, stroma targeting by OVs demands some caution, as it could have major effects on the tumor composition, immune infiltration and activation, as well as off-target effects on surrounding cells. Altogether, by taking into account the complex interactions between the heterogenous stroma components and tumor cells, OVs and their arming transgenes hold promise as unique dual tumor- and stroma-targeted therapies that could ultimately improve the prognosis of cancer patients, especially those suffering from stroma-rich tumors.


TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 1 and 2 (TCP) family proteins are the plant-specific transcription factors extensively participating in diverse developmental processes by integrating external cues with internal signals. The roles of CINCINNATA (CIN)-like TCPs are conserved in control of the morphology and size of leaves, petal development, trichome formation and plant flowering. The tight regulation of CIN-like TCP activity at transcriptional and post-transcriptional levels are central for plant developmental plasticity in response to the ever-changing environmental conditions. In this review, we summarize recent progresses with regard to the function and regulation of CIN-like TCPs. CIN-like TCPs are regulated by abiotic and biotic cues including light, temperature and pathogens. They are also finely controlled by microRNA319 (miRNA319), chromatin remodeling complexes and auxin homeostasis. The protein degradation plays critical roles in tightly controlling the activity of CIN-like TCPs as well.  Developmental plasticity is central for sessile plants in adaptation to the environmental conditions . The molTEOSINTE BRANCHED1 (TB1) from maize (Zea mays) . Bof ARR16  115]. T. TBRAHMAtie1-D obtained by screening a collection of activation tagging mutants for leaf-defective ones. Overexpression of TIE1 in tie1-D or in transgenic plants using CaMV 35S promoter to drive TIE1 all led to curled and serrated leaves that are observed in the multiple cin-like tcp mutants . T. T116]. elopment  [116]. Iranching . The funirsutum) . GhTIE1 ranching . A similranching . SPL/NZZike TCPs . Overexp mutants . These rTEAR1 leads to serrated and curled leaves similar to that observed in the multiple cin-like tcp mutants and tie1-D [TEAR1 indirectly regulates the activity of CIN-like TCPs by switching the interactors of CIN-like TCPs from TIE1 to BRM /INDOLE-3-ACETIC ACID (IAA) repressors mediate auxin signaling by recruiting TPL/TPRs to suppress the activity of AUXIN RESPONSE FACTORS (ARFs) ,120,121.d tie1-D . These f1 to BRM , thus chCIN-like TCPs are dynamically and specifically expressed in organs and also are induced by environmental signals including light and temperature [CIN-like TCP genes are insufficient. The transcriptional repressor RABBIT EARS (RBE) has been reported to decreased the expression of TCP4, TCP5, TCP13 and TCP17 in promoting petal growth and TCP4 and TCP5 are possibly direct targets of RBE [CIN-like TCPs are necessary for elucidating other upstream regulators, especially the direct regulators. The truncated promoters can be used to drive reporters in determining the minimal regions required for the expression patterns of CIN-like TCPs. Transcription factors directly interacting with the promoters of CIN-like TCPs could be identified by yeast-one-hybrid screening. CIN-like TCPs are key transcription factors essential for plant growth and development in response to environmental cues and internal signals. The temporal and spatial activity of CIN-like TCPs determines cell proliferation, expansion and differentiation of cells in different organs in shaping plant morphology at various developmental stages. Consequently, the fine-tuning of CIN-like TCP activity is critical for plant developmental plasticity. At the transcriptional level, perature ,92,95. Hs of RBE ,71. MoreBRC1 belonging to CYC/TB1-like TCP group of class II TCPs [IAA CARBOXYL METHYLTRANSFERASE1 (IAMT1) which converted IAA to methyl-IAA ester led to curly leaves and reduced the expression level of some CIN-like TCPs [CIN-like TCPs at the transcriptional level. Further studies are needed to determine whether other plant hormones and environmental signals except light and temperature could possibly regulate CIN-like TCPs and how these signals could be integrated to control the activity of CIN-like TCPs.CIN-like TCPs are central for regulating biosynthesis and signaling of different phytohormones including auxin, JA and brassinosteroid (BR) ,123,124. II TCPs ,125,126.ike TCPs , indicatTCP regulation module is conserved and widely studied in several plant species [CIN-like TCPs exist in different plant species? which are those transcription factors deciding the expression level and pattern of MIR319 genes? These questions are still open.At the post-transcriptional level, the miR319- species ,63. CoulArabidopsis and the other plant species.At the protein level, we know little about the degradation mechanisms of CIN-like TCPs mediated by 26S proteasome or other protein degradation pathways. The regulation mechanisms of CIN-like TCPs by class I TCP transcription factors and other interacting proteins are still largely unknown. It is still a challenge to thoroughly understand the shaping of plant morphology controlled by the CIN-like TCP-centered network under various environmental and developmental conditions in


Science by Shrock et al.1 examined how responses to COVID-19 severity differed amongst patients with different prior viral exposure history, finding notable correlations between both. Such serological profiling provides a window into differential viral responses amongst patients with diverse outcomes, potentially mediating improved therapeutics and vaccines for SARS-CoV-2.1A recent study published in 2The ongoing global coronavirus disease-2019 (COVID-19) pandemic caused by the novel respiratory coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlies widespread global morbidity, but particularly rapidly overwhelmed medical facilities of Europe and North America. Indeed, only 9% of deaths have occurred in Asia where the outbreak originated, while Europe and North America account for 75% of case fatalities.1 examined the antibody profiles of 232 COVID-19 patients and 190 pre-COVID-19 era controls, identifying several antibody epitopes shared between other coronaviruses (CoVs) and SARS-CoV-2. COVID-19 patients requiring hospitalization. Such patients were also observed to exhibit stronger and broader antibody responses to SARS-CoV-2, but weaker responses to past infections compared with those who did not need hospitalization.1 This perhaps suggests that patients who did not need hospitalization had exhibited a stronger response to previous infections.Shrock et al.1 However, this could also just be indicative of a lower immune capacity as opposed to acquired immunity through multiple infections. Astoundingly though, in the same study, Shrock et al.1 also observed that COVID-19 patients requiring hospitalization exhibited a higher seroprevalence rate for cytomegalovirus (CMV) and herpes simplex virus 1 (HSV-1), indicating that even though exposure to non-CoVs mounted an adequate immune response, this was unable to prevent hospitalization following SARS-CoV-2 infection. Conversely, the hospitalized group of COVID-19 patients exhibited a lower response to CMV and HSV-1 peptides, suggesting a reduced immune capacity.1Perhaps the stronger the exposure/response to previous CoV infections, the lower the chances that hospitalization was required upon SARS-CoV-2 infection. Indeed, a significantly lower level of antibodies targeting common viruses including rhinoviruses, enteroviruses, and influenza was observed in COVID-19 patients requiring hospitalization compared to those who did not.2 All CoVs consist of transmembrane trimeric spike (S) glycoproteins, membrane (M) glycoproteins, and transmembrane envelope (E) proteins. The RNA of CoV is bound to nucleocapsid (N) proteins resembling string-on-beads. Due to high levels of homology between these proteins in SARS-CoV, MERS-CoV, and SARS-CoV-2, perhaps prior exposure to one virus could confer partial immunity to another. Thus, perhaps an ‘adaptive immune response’ due to repeated/increased CoV exposure in Asian/Middle Eastern populations underlies low morbidity seen in those regions.2Seven CoVs have been associated with human disease, mostly amounting to mild respiratory illness, perhaps underlying 15–30% of global common colds. Previous CoV outbreaks have included severe respiratory syndrome (SARS) caused by SARS-CoV, and Middle East respiratory syndrome (MERS) caused by MERS-CoV. Significantly, SARS-CoV, MERS-CoV, and SARS-CoV-2 share significant sequence homology and antigenic epitopes capable of inducing an adaptive immune response.1 indicated that peptides corresponding to the S and N proteins were most reactive in COVID-19 patient sera, but significantly lower in pre-COVID era controls. However, the third most frequently recognized peptide in COVID-19 patient sera was the replicase polyprotein ORF1, recognized to a similar degree in pre-COVID era control sera as well. This was posited to be likely due to cross-reactivity between antibodies elicited by previous CoVs exposure, suggesting that such patterns represented a pre-existing cross-reactive response.1 Indeed, mapping highly conserved regions of the S protein between SARS-CoV-2 and other common CoVs, as well as specific regions mapping to individual CoVs, indicated cross-reactivity with SARS-CoV, MERS-CoV, and seasonal CoVs to varying degrees. This phenomenon, perhaps, could potentially be attributed to antibody production induced by other pathogens. Collectively, such data seems to suggest that responses to seasonal CoVs may be able to influence the immune response to SARS-CoV-2.1Shrock et al.2 Indeed, prior viral exposure could provide some protection if cross-reactive neutralizing antibodies or T cell responses are stimulated upon exposure to SARS-CoV-21  of live attenuated vaccines such as the Bacilli-Calmette-Guerin (BCG) and Polio (OPV) vaccines.5 The thought is that such vaccines broadly stimulate the innate immune system to confer non-specific protection against diseases other than the intended target of that vaccine, in essence ‘training’ components of the innate immune system.5 While the exact measurement of such protection provided remains unclear, numerous data suggest that at least some level of immunity is provided, with quantifiable reductions in mortality. Perhaps in such cases, a similar mechanism of prior ‘molecular immunity training’ is at play as observed by Shrock et al.1Such potential mechanisms whereby patients are rendered with levels of ‘antibody memory’ against various prior viral infections could thus be utilized in both therapeutic and vaccinology applications. One such demonstrative example is that of intravenous immunoglobulin (IVIG) administration, which through formation of antigen–antibody complexes and their neutralization, mitigating autoimmune responses.1Regardless, serological profiling has provided a window into viral responses amongst patients with diverse outcomes. In particular, we are only just beginning to understand how prior viral exposures may influence current/future responses. Of course, demographic and socioeconomic factors make it difficult to draw strong conclusions, and it will be some time before a suitable patient population can be examined to definitively obtain such answers. However, studies such as the one performed by Shrock et al. provide significant stepping-stones in understanding and isolating the molecular mechanisms of SARS-CoV-2 and COVID-19. Collectively, such milestones will potentially enable us to inform the production of improved diagnostics, therapeutics, and even vaccinations for SARS-CoV-2.Permission to use modified figure


One out of seven patients with severe aortic stenosis (AS) undergoing transcatheter aortic valve replacement (TAVR) may be affected by transthyretin cardiac amyloidosis (ATTR-CA), mostly presenting with low-flow low-gradient AS with mildly reduced ejection fraction. The complex interaction of these two pathologies poses specific diagnostic and management challenges. The prognostic implications of this clinical intersection are not defined yet. Moreover, whether TAVR may have a prognostic benefit in ATTR-CA patients with symptomatic severe AS remains unclear, posing doubts on the best management strategy in this increasingly recognized subset of patients.We present a case of an 87-year old man with low-flow low-gradient severe AS, for whom a diagnosis of ATTR-CA was suspected based on clinical and echocardiographic criteria specific to coexisting AS and ATTR-CA. The diagnosis was eventually confirmed by positive bone tracer scintigraphy imaging. Following in-depth Heart team discussion, integrating frailty and prognostic information from combined cardiomyopathy states, a decision was made to manage the patient’s severe AS conservatively.In the presented case, we deemed the natural history of ATTR-CA amyloidosis to negatively affect both the patient’ prognosis and procedural risk, adversing TAVR indication despite symptomatic severe AS. No clear evidence is currently available to guide decision making in this setting, advocating for prospective studies to clarify if TAVR may have a prognostic benefit in ATTR-CA - and which ATTR-CA - patients. One out of seven patients with severe aortic stenosis (AS) undergoing transcatheter aortic valve replacement (TAVR) may be affected by transthyretin cardiac amyloidosis (ATTR-CA), mostly presenting with low-flow low-gradient AS with mildly reduced ejection fraction . The comA 87-year old man with a presenting diagnosis of hypertensive cardiomyopathy, orthostatic hypotension, moderate aortic stenosis, permanent atrial fibrillation, left bundle branch block and preserved ejection fraction (EF)  was admitted to our Clinic with Canadian Cardiovascular Society III angina, New York Heart Association (NYHA) III functional class and signs of pulmonary and systemic venous congestion Fig. .Fig. 1a2) with severe concentric remodeling , mildly reduced EF (50%), severely depressed global longitudinal strain (− 3.1%) with no relative apical sparing pattern and an average mitral annular Tissue Doppler S′ of 5.5 cm/sec. Second degree diastolic disfunction and severe biatrial dilatation were also apparent. A low QRS voltage-to-LV mass ratio by Sokolow-Lyon criteria was observed. Aortic valve evaluation revealed low flow-low gradient severe aortic stenosis (AS) , confirmed by computed tomography scan .Cardiac ultrasound documented left ventricle (LV) hypertrophy .Based on the low-flow low-gradient AS phenotype in the background of severe concentric hypertrophy, biatrial dilatation, S′ wave depression, conduction abnormalities, low QRS-voltage-to-LV-mass ratio and history of orthostatic hypotension, a diagnosis of amyloid cardiomyopathy was suspected. Bone tracer scintigraphy was performed revealing high diffuse myocardial uptake (Perugini score 3), diagnostic - in the context of an absent monoclonal protein on serum and urine exams  - for transthyretin cardiac amyloidosis (ATTR-CA)  The patient was unsuitable for ATTR-CA specific treatment because of current national reimbursement policies.A Heart Team evaluation was carried. In consideration of the high procedural risk (EuroSCORE II 11.4%) transcatheter aortic valve replacement (TAVR) appeared indicated for severe AS treatment. However, considering the patient frailty , hypoalbuminemia (1 point), lower-extremity muscle weakness (2 points) ), the prATTR-CA is a common under recognized condition among patients with severe AS, mostly presenting with low-flow low-gradient AS with mildly reduced EF . A high In this context, no solid evidence is currently available to inform the diagnostic strategies for ATTR-CA among patients with severe AS and, more importantly, whether and in which ATTR-CA patients TAVR (and which with TAVR device ) may havIn the presented case, a comprehensive heart team evaluation deemed the natural history of ATTR-CA to negatively affect both the patient’s prognosis and procedural risk, leading, along with consideration of patient preferences, to a lack of indication to TAVR during the index hospitalization despite severe AS. At mid-term follow-up, the patient was clinically stable without ensuing decompensation episodes, accordingly, clinical follow-up is ongoing. This management strategy, despite based on objective functional, laboratoristic and imaging parameters along with patient preferences, was based on a subjective weight of the numerous elements taken into considerations, as solid data regarding risk stratification and management of severe AS in patients with ATTR-CA are currently lacking. Indeed, no clear guidance to orientate decision making in this setting is available, advocating for prospective studies to clarify if TAVR may have a prognostic benefit in ATTR-CA - and which ATTR-CA-patients.


Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms  were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in  X. citri subsp. citri (Xcc) with symptoms of water-soaked eruptions, circular lesions, and pustule-like lesions on all plant tissues, resulting in heavy economic losses ; (4) target sequence interference; and (5) highly homologous within family [–24 nt;  RT-qPCR, but not with the results derived from stem–loop RT-qPCR [Moreover, the efficiency of PCR amplification for the stem–loop primer was lower than the poly-A tail, and some of them were even below 90%. Accordingly, the poly-A tail qPCR has higher amplification efficiency and better sensitivity than the stem–loop RT-qPCR. In forensic casework, poly-A tail extension also exhibited apparently more amplification than stem–loop RT for a body fluid identification of miR-451 and miR-205 . In addicroarray . Similar RT-qPCR .C. sinensis and C. reticulata infected with a citrus canker pathogen, five algorithms: Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder were used for screening reference genes. Poly(A) extension RT-qPCR and stem–loop RT-qPCR were performed to determine the most appropriate method for detecting expression patterns of miRNA. Our results showed that poly(A) RT-qPCR is a more reliable method than stem–loop RT-qPCR, and miR162-3p and miR472 genes in poly(A) RT-qPCR are the most stable internal control genes for miRNA profiling analysis of citrus infected by Xcc. The selection of reference genes is critically important for the accurate quantification of target gene expression in RT-qPCR analysis. To identify appropriate miRNA reference genes for normalizing the level of miRNA expression in