Oncolytic viruses represent interesting anti-cancer agents with high tumor selectivity and immune stimulatory potential. The present review provides an update of the molecular mechanisms of the anti-neoplastic and immune stimulatory properties of the avian paramyxovirus, Newcastle Disease Virus (NDV). The anti-neoplastic activities of NDV include (i) the endocytic targeting of the GTPase Rac1 in Ras-transformed human tumorigenic cells; (ii) the switch from cellular protein to viral protein synthesis and the induction of autophagy mediated by viral nucleoprotein NP; (iii) the virus replication mediated by viral RNA polymerase (large protein (L), associated with phosphoprotein (P)); (iv) the facilitation of NDV spread in tumors via the membrane budding of the virus progeny with the help of matrix protein (M) and fusion protein (F); and (v) the oncolysis via apoptosis, necroptosis, pyroptosis, or ferroptosis associated with immunogenic cell death. A special property of this oncolytic virus consists of its potential for breaking therapy resistance in human cancer cells. Eight examples of this important property are presented and explained. In healthy human cells, NDV infection activates the RIG-MAVs immune signaling pathway and establishes an anti-viral state based on a strong and uninhibited interferon α,ß response. The review also describes the molecular determinants and mechanisms of the NDV-mediated immune stimulatory effects, in which the viral hemagglutinin-neuraminidase (HN) protein plays a prominent role. The six viral proteins provide oncolytic NDV with a special profile in the treatment of cancer. An OV can be an attenuated naturally existing virus, or it can be a genetically engineered recombinant virus. Four OVs are presently approved for some clinical cancer treatment: Oncorine H101), Rigvir, T-VEC . A network of 24 TFs was predicted to regulate 779 of the 1351 upregulated genes. The effect of NDV on mDCs was highly reproducible. The timing of this step-wise transcriptional signal propagation appeared as highly conserved .The NDV infection of DCs activates NFκB, just like in NK cells and macrophages. This TF induces in DCs a module for the expression of pro-inflammatory cytokines together with a module for antigen presentation. These modules promote immunogenic DC1 differentiation with the potential to stimulate CD4+ T helper 1 responses and CD8+ effector T cells . CytokinR from IOZK  [Antigen cross-presentation to CD8+ T cells is a specialized function conducted predominantly by DCs. Transporter associated with antigen processing (TAP) dysfunction in DCs enables noncanonical cross-presentation for T cell priming . OncolysGermany) ,114.DCs pulsed with patient-derived NDV oncolysate were reported almost 20 years ago to potently stimulate autologous cancer-reactive memory T cells. Memory T cells from the bone marrow, activated in this way, secreted increased amounts of IFN-α and IL-15  ligands DC maturation can be induced not only by native NDV but also by NDV-derived virus-like particles (VLPs). Such VLPs can induce DC maturation through the TLR4/NFκB pathway. This facilitates DC migration via the CCR7-CCL19/CCL21 chemokine axis . The rouNaïve T cells are maintained in a quiescent state that promotes their survival and persistence . AntigenHN molecules at the surface of NDV-infected tumor cells introduce new cell adhesive strength for interaction with lymphocytes and for the costimulation of T cells . HN tetrAntigen peptide-presenting cells, stably transfected with HN c-DNA from NDV, caused a six-fold increase in peptide-specific CD8+ CTL responses, compared to untransfected antigen peptide presenting cells . These fThe immunization of mice with NDV-infected tumor cells was demonstrated (i) to strongly increase tumor cell immunogenicity, (ii) to augment cooperative interactions between tumor-specific CD4+ Th and CD8+ CTLPs ,91, and Studies with human cells revealed that the NDV infection of tumor cells could break T-cell tolerance of a melanoma specific Th cell clone. This was due to the introduction of a CD80/CD86 independent co-stimulatory activity .These examples demonstrate the T cell co-stimulatory activity of NDV for mouse CD8+T cells and for human CD4+ T cells. The increase in adhesive strength and costimulation could contribute to breaking T-cell tolerance to TAAs. T-cell tolerance towards TAAs and T-cell exhaustion are phenomena often observed in late-stage disease with a chronic excess of TAAs.HN plasmid DNA was demonstrated to induce IFN-α and to be a powerful molecular adjuvant for anti-tumor vaccination . Co-exprThe above findings demonstrate that the HN molecules of NDV play an important role in the induction of an IFN-I response and that the IFN-I response in combination with an anti-TA response facilitates the induction and activation of a proper TAA-specific CD8+ CTL response, targeting the site of the tumor.CTLs and NK cells eliminate infected cells or tumor cells by triggering apoptosis. The contents of lytic granules of killer cells, such as perforins  and granAfter the extensive proliferation and execution of effector programs within the first two weeks after antigen stimulation, a subset of these T cells becomes quiescent and develops into memory T cells . The mecVirus potentiation of tumor vaccine T cell stimulatory capacity was reported to require cell surface binding but not infection . This caIn a human melanoma nude mouse xenotransplant model, strong anti-tumor bystander effects were observed by a 20% co-admixture of melanoma cells pre-infected in vitro by lentogenic NDV (Ulster), suggesting oncolysis-independent innate immunity activation .Oncolysis-independent effects are included in Summary: Cellular and molecular mechanisms involved in NDV-mediated immune stimulatory activity-Increase in adhesive interactions between infected tumor cells and immune cells .-Activation of innate immunity cells: (i) signaling via HN-NKp44/46 in NK cells and (ii) NFkB mediated upregulation of TRAIL, secretion of TNFa and NO in monocytes and macrophages, and induction of a module for antigen presentation in DCs.-Reprogramming of DCs to DC1 by a choreographed cascade of transcription factors.-Oncolysate uptake by DCs and promotion of antigen cross-presentation.-Oncolysis-independent immune stimulation with pro-inflammatory and abscopal effects.-Augmentation of tumor cell immunogenicity.-Augmentation of CD8+ T cell costimulation.-Breakage of CD4+ T cell tolerance to tumor-associated antigens (TAAs).-Augmentation of cooperative interactions between CD4+ T helper and CD8+ cytolytic T cell precursors (CTLPs).-Increase in frequencies of CD4+ and CD8+ CTLPs via induction of interferon a/ß.-Induction of immunogenic cell death (ICD) with expression of PAMPs  and release of DAMPs .-Cognate interaction of DC1 TAA-presenting cells with CD4+ T cells leading to TA-specific activation and Th1 polarization.-Th1 CD4+ T cells interacting with CD8+ T cells helping their differentiation into CTLs and CD8+ memory T cells.-R) activating similar mechanisms in patients leading to long-term T cell mediated immune memory.Patient-derived NDV-modified cancer vaccines , as well as resistance to targeted therapies, apoptosis, TRAIL, and hypoxia. Of immunological relevance is that oncolytic NDV can break T-cell tolerance to TAs. This has to do with the HN-mediated improvement of adhesiveness between tumor cells and T cells and its co-stimulatory potential.Other OVs also target oncogenic pathways. For example, activating mutations in the small GTPase Ras block PKR. This process can facilitate the selective replication of reovirus, HSV-1, adenovirus, and vaccinia virus .NDV infection of cancer cells activates PI3K/Akt/mTOR, p38 MAPK/Mnk1, and CDK-Rb-E2F oncogenic pathways which help viral mRNA translation. Of importance for the understanding of tumor-selective virus replication and oncolysis are also the recent findings about the induction of ER stress and UPR signaling. NDV activates all three branches of UPR signaling: PERK-eIF2α, ATF6, and IRE1α. Signaling via these intrinsic pathways induces autophagy, apoptosis, and tumor-selective oncolysis. Distinct NDV proteins  have been implicated in the tumor-selective induction of autophagy. Autophagy facilitates a switch from cellular to viral protein synthesis, antigenome self-assembly and viral replication in infected tumor cells. In addition to the intrinsic pathways of cell death, the induction of ICD causes activation of immune cells, which initiate the extrinsic mechanisms of cell death.Structural studies of NDV viral proteins revealed various levels of assembly potential: NP monomers forming helical clam-shaped nucleocapsid structures and M proteins assembling in host cell plasma membranes into hollow helical oligomers; HN proteins on infected tumor cell membranes forming dimers and tetramers capable of interacting with NK cells and T cells, thereby cross-linking activating receptors; and molecular interactions between HN and F proteins, leading to fusion promotion activity of importance for virus cell entry, syncytium formation, and virus spread.NDV can infect cells by membrane fusion, thus entering its genome directly into the cell’s cytoplasm where the viral RNA can activate PKR and RIG-I. In human non-transformed cells, such as lymphocytes, NDV induces in this way a strong IFN-I response, which prevents viral replication and leads to fast virus clearance. The strong IFN-I response protects infected cells in an autocrine way and uninfected cells in a paracrine way. While NDV and other virus-derived RNAs mediate crucial signals for innate immunity via the RIG-I-MAVS and IFNAR/JAK-STAT pathways, pathogen-derived DNA mediates respective signals via the cGAS-STING pathway .In addition to its anti-neoplastic activities, NDV has immune stimulatory properties. The viral HN protein causes activation of NK cells by direct molecular interaction with the cytotoxicity-activating receptors NKp46 and NKp44. pHN plasmid DNA stimulates the production of IFN-I and can be used as a powerful vaccine adjuvant. Analysis of the induction requirements for T-cell-mediated protective anti-tumor immunity in an animal model revealed that type I IFNs play an important role in the augmentation of an anti-tumoral CD8+ CTL response.Human dendritic cell activation by NDV can serve as a paradigm for the virus’s immune stimulatory properties. The cellular response is not inhibited by any viral escape mechanism. The establishment of an anti-viral state of DCs occurs within 18 h and involves in a choreographed way more than 24 TF proteins. One of these, NFκB, induces a module for antigen presentation and a module for the expression of pro-inflammatory cytokines for DC1 differentiation.NDV oncolysate is taken up by DCs via macropinocytosis and via DNGR-1 receptor-mediated endocytosis. This leads to antigen processing and presentation via MHC II to CD4+ T cells and to antigen cross-presentation via MHC I to CD8+ T cells. Cooperative CD4+ and CD8+ T cell interactions, facilitated by NDV-induced IFN-I, then lead to the establishment of tumor-specific adaptive immune reactivity and systemic protective immunological memory.Pre-clinical studies in animal models and progress in immunological basic research provide new insights into in vivo mechanisms ,94. For Oncolytic NDV has been applied to cancer patients for more than 55 years and clinical effects have been described in several reviews ,156,157.Evidence for the clinical utility and effectivity of NDV and NDV-derived cancer vaccines has been obtained in many experimental studies, including Phase I/II studies and one prospective randomized Phase II/III study. Further clinical trials are ongoing.To conclude, OVs like NDV can target oncogenic pathways and exploit cancer immune evasion pathways and can induce local and systemic anti-tumor immunity. To perform such complex activities, oncolytic NDV makes use of all six of its structural proteins. Molecular analyses revealed that oncolytic NDV (i) can use multiple ways of cell entry, (ii) can interfere with multiple cancer-typical signaling pathways, (iii) can use multiple branches of UPR signaling, (iv) can kill cancer cells in multiple ways, (v) can use multiple ways of breaking therapy resistance, (vi) can activate immune cells in multiple ways, and (vii) can activate multiple interferon regulatory factors . Oncolytic NDV thus appears to have various options on how to infect and destroy human tumor cells. This explains the broad range of tumor cell types being susceptible to this OV: carcinoma, melanoma, GBM, lymphoma, etc.With all the mentioned intrinsic activities, oncolytic NDV can be considered a potent platform for further modifications. One example of such a vaccine platform is a recently published NDV vector-based SARS-CoV-2 vaccine candidate. A study in 62 piglets revealed safety and immunogenicity upon intranasal application to induce mucosal immunity and upon intramuscular application. The vaccine can be produced at low cost in embryonated eggs in established facilities that are used to produce influenza virus vaccines .Advances in the study of antitumor immunotherapy via NDV have recently been reviewed ,161,162.Some of the oncolytic and immune stimulatory properties of NDV are shared by other OVs ,161,162.So far, studies are lacking concerning the optimum delivery and dosage for maximal efficacy of oncolytic NDV. Further research is necessary to elucidate the mechanisms of NDV spread in tumors, to improve the efficacy of systemic tumor targeting, and to unravel the effects of NDV on cancer stem cells. Of particular interest is the combination of oncolytic NDV with immune checkpoint blockade therapy because synergistic effects are reported from pre-clinical studies.OV therapy resistance is another problem of relevance. New biomarkers may help to elucidate molecular mechanisms of resistance and to predict OV therapy success . Small mOf great interest should be research to evaluate the potential of oncolytic NDV in targeting the many downstream pathways controlled by Rac1 and to compare this with SMI leads in cancer drug development . The facAt times of personalized and individualized medicine the concept of randomized-controlled clinical trials (RCTs) as exerted by big pharma companies in the last decades is being questioned and should be further discussed . A compaThe therapeutic potential of NDV can be further augmented by the incorporation of therapeutic transgenes ,168 and/Future improvements of clinical results in late-stage cancer patients can be expected by combining OV therapy with adoptive T cell therapy.Three main questions concerning oncolytic NDV and its mechanism of function were dealt with in this review. 1. How can the virus efficiently replicate in a tumor cell undergoing oncolytic cell death with a shut-off of cellular protein synthesis? 2. How can infected non-transformed human cells prevent virus replication? 3. How can different types of immune cells become activated by this avian paramyxovirus? According to latest and previous milestone findings the answers provided are: 1. Following NDV-induced PKR-eIF2α-mediated host protein shut-off in human cancer cells, the viral nucleoprotein NP binds to the cellular eIF4E protein within the mRNA translation initiation complex and directs translation exclusively towards viral proteins. In addition, the NP protein induces autophagy, protects the viral antigenome against degradation, and facilitates virus replication. 2. In non-transformed human cells, the signaling pathways induced by the interaction of viral pppRNA Leader with RIG-I and by dsRNA with PKR induce a strong IFN-I response, establish an antiviral state, and prevent viral antigenome nucleocapsid complex formation. 3. Innate immune cells become activated by NDV via NFκB and are re-programmed via distinct networks of transcription factors towards their specific effector functions. In the case of dendritic cells, this includes programs of antigen cross-presentation and of activation of pro-inflammatory cytokines. The viral HN protein activates NK cells via binding to NKp46 and activates IFN-I responses and co-stimulates T cells. Viral oncolysis causes release of TAs, PAMPs, DAMPs, and cytokines. This immune stimulatory environment leads to the generation of cancer-specific adaptive immune responses.


Obesity and type 2 diabetes mellitus (T2DM) are highly prevalent disorders, associated with insulin resistance and chronic inflammation. The brain is key for energy homeostasis and contains many insulin receptors. Microglia, the resident brain immune cells, are known to express insulin receptors (InsR) and to be activated by a hypercaloric environment. The aim of this study was to evaluate whether microglial insulin signaling is involved in the control of systemic energy homeostasis and whether this function is sex-dependent. We generated a microglia-specific knockout of the InsR gene in male and female mice and exposed them to control or obesogenic dietary conditions. Following 10 weeks of diet exposure, we evaluated insulin tolerance, energy metabolism, microglial morphology and phagocytic function, and neuronal populations. Lack of microglial InsR resulted in increased plasma insulin levels and insulin resistance in obese female mice. In the brain, loss of microglial InsR led to a decrease in microglial primary projections in both male and female mice, irrespective of the diet. In addition, in obese male mice lacking microglial InsR the number of proopiomelanocortin neurons was decreased, compared to control diet, while no differences were observed in female mice. Our results demonstrate a sex-dependent effect of microglial InsR-signaling in physiology and obesity, and stress the importance of a heterogeneous approach in the study of diseases such as obesity and T2DM. Obesity and type 2 diabetes mellitus (T2DM) are global chronic disorders mainly caused by a disbalance between food intake and energy expenditure . In the A key factor known to disrupt energy homeostasis and cause obesity is a hypercaloric diet . ObesityWe hypothesized that reduced microglial insulin signaling affects the microglial immune function, which will ultimately result in insufficient maintenance of a healthy brain environment and neuronal dysfunction. To test our hypothesis, we generated mice with a microglia-specific knockout of the InsR gene. The aim of this study was to evaluate whether microglial insulin signaling is involved in the control of systemic energy homeostasis. Since it has been shown that estrogen may have a protective effect on the development of insulin resistance, diabetes, and obesity, both in humans and animals , we perffl/fl) were crossed with mice carrying tamoxifen-inducible Cre recombinase, driven by the Cx3cr1 promoter (Cx3Cr1CreERT2)  in vitro. Primary microglia from Insrwt/wt—Cx3Cr1CreERT2 were used as controls (InsR-Ctrl). All primary microglial cells were treated with 4-hydroxytamoxifen to delete the Insr gene. We confirmed a significant decrease of InsR protein in InsR-KO microglia compared to InsR-Ctrl  A. We usensR-Ctrl B,C.To understand the importance of microglial InsR function in vivo, we induced the knock-out as mentioned above at the adult stage of mice. Both male and female mice were treated with tamoxifen at 6 weeks of age to generate microglial InsR-Ctrl and InsR-KO animals. At 8 weeks of age, mice were fed either a Chow diet, representing the physiological dietary environment or high-fat diet (HFD), known to induce obesity, for the duration of 12 weeks. Both male and female microglial InsR-KO mice, fed an HFD, showed increased caloric intake at the end of 12 weeks of HFD exposure when compared to HFD-fed InsR-Ctrl mice D,F. InteWhen evaluating the effect of microglial loss of InsR function in females on body weight, we found no significant differences in the BW and BW gain between InsR-Ctrl and InsR-KO mice on the Chow diet. However, we observed a strong trend for increased BW and BW gain from week 3 onwards in obese InsR-KO females, compared to InsR-Ctrl H,I. In mWhile evaluating the effect of microglial InsR-KO on adiposity, we found that Chow-fed InsR-Ctrl female mice had lower perirenal white adipose tissue (WAT), compared to InsR-KO mice on Chow diet, as well as compared to InsR-Ctrl, HFD-fed mice L. No difThese data suggest that loss of microglial InsR may play a role in the progression of obesity in a sex-dependent manner.Next, we evaluated the effect of microglial insulin function on glycemic homeostasis and insulin sensitivity in both control and obesogenic conditions. Following 11 weeks of obesogenic diet exposure, we observed a decrease in fasting glucose in InsR-KO female mice compared to InsR-Ctrl A. MoreovIn males, InsR-KO mice had higher basal blood glucose compared to InsR-Ctrl mice one week following tamoxifen injection . HoweverThese data confirm our sex-dependent observations and suggest a key role of microglial insulin signaling in glucose homeostasis in female mice during obesity.To further investigate the implications of lack of microglial InsR signaling in physiological and obesogenic conditions, we evaluated different metabolic parameters following chronic exposure to control or hypercaloric diet.Female InsR-KO mice had a lower respiratory exchange rate (RER) during the dark (active) phase when compared to InsR-Ctrl under obesogenic conditions E,F, indiIn males, no differences in RER were observed between InsR-KO and InsR-Ctrl mice, irrespective of the dietary intake H,I. InsRThese data suggest that loss of InsR in obese female mice leads to higher reliance on fat metabolism during the active phase of the animals.Hcrt, Agrp, and Npy gene expression between InsR-KO and InsR-Ctrl animals, irrespective of sex or diet , agouti-related peptide (Agrp), and neuropeptide Y (Npy). We observed no changes in  or diet A–F. We fCd68), a commonly used phagocytic indicator in Iba1-ir microglia; Interleukin-1 beta (Il1b) cytokine, highly produced by microglia; and inhibitor of nuclear factor-kappa b kinase subunit beta (Ikbkb), kinase involved in the phosphorylation of the inhibitor/NFkB complex, thus activating the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), involved in an inflammatory response. We found an increase in Cd68 gene expression in female InsR-KO mice compared to InsR-Ctrl mice under obesogenic conditions , a key brain nucleus involved in systemic control of energy metabolism. Microglial cell number and morphology were evaluated by ionized calcium-binding adaptor molecule 1 (Iba1) immunoreactivity. We found a borderline significant decrease of microglial cell number in InsR-KO female mice, compared to InsR-Ctrl during obesogenic conditions A,B. In mCd68 gene expression in the whole hypothalamus of female mice, next we evaluated the phagocytic capacity of Iba1-ir cells specifically in the ARC. We evaluated the immunoreactivity of CD68 in Iba1-ir microglia. We found higher CD68-ir in microglia of InsR-KO female mice on Chow diet when compared to InsR-Ctrl mice  neurons, as they have been previously shown to be highly sensitive to a chronic hypercaloric environment . We evalIn this study, we showed that microglial insulin signaling plays a sex-dependent role in the control of systemic energy homeostasis, with microglial InsR being involved in the progression of obesity most prominently in female mice. We found a decrease in primary microglial projections in all InsR-KO animals compared to InsR-Ctrl animals, irrespective of diet or sex. In obese female mice, loss of microglial InsR resulted in decreased microglial cell numbers, higher fasting plasma insulin, and insulin resistance. Therefore, we propose that microglial insulin signaling plays a key protective role during the progression of obesity in female mice.The decrease in the primary microglial projections in all microglia-InsR-KO animals, when compared to microglia-InsR-Ctrl animals, is in line with the study of García-Cáceres et al., who reported that astrocytes lacking insulin receptors have fewer and shorter primary projections when compared to control astrocytes . In addiDue to the important role of the hypothalamus, and specifically the ARC nucleus in energy metabolism, we decided to study the effect of microglial insulin receptor loss on the anorexigenic population of POMC neurons. We observed no changes in POMC neurons in females, while there was a significant loss of POMC neurons in microglia-InsR-KO male mice, compared to microglia-InsR-Ctrl in obesity. This sex-specific decrease of POMC neurons probably can be explained by the decrease of phagocytic capacity we found in microglia-InsR-KO males in obesity, as previously it has been demonstrated that impaired microglial phagocytic capacity leads to loss of POMC neurons ,38. AddiUp until recently, rodent studies on metabolic disorders mainly focused on males and sex differences have been understudied, especially concerning microglia. However, emerging data are pointing to a strong heterogeneity of microglia between males and females ,41. A stIn summary, the findings of this study highlight the importance of microglial insulin signaling in the CNS control of energy metabolism and the sex-dependent differences observed during the progression of obesity. Microglial cells seem to show a vast difference in their phenotype between males and females in an age-, region-, and disease-dependent manner ,41,44. Ofl/fl mice  [CreERT2 mice , which express the tamoxifen-inducible Cre recombinase, driven by the fractalkine chemokine (C-X3-C motif) receptor 1 (Cx3Cr1) promoter [fl/fl-Cx3Cr1CreERT2 mice are referred to from now onwards as InsR-KO animals. Their littermates, InsRwt/wt-Cx3Cr1CreERT2 mice, are referred to as InsR-WT animals (controls). All studies were approved by the Animal Ethics Committee of the Royal Dutch Academy of Arts and Sciences  and performed according to the guidelines on animal experimentation of the Netherlands Institute for Neuroscience .Microglia-specific postnatal InsR-KO male and female mice were generated by crossing InsR5, JAX®)  with Cx3promoter . All micwt/wt-Cx3Cr1CreERT2 and InsRfl/fl-Cx3Cr1CreERT2 mouse brains. Briefly, brains were cultured until 80–90% confluency of mixed glial culture, when cells were provided with L929 cell line-conditioned medium containing colony-stimulating factor-1 (CSF1) to promote microglial proliferation. Microglial cells were collected and seeded for evaluation of knock-out efficiency. All cells were treated with a culture medium containing 1 µM 4-hydroxytamoxifen for 48 h and further used for evaluation of InsR protein.Primary microglial cells were isolated from neonatal InsRIsolated primary microglial samples (see above) were used for evaluation of knock-out efficiency by measuring InsR protein concentration, following the established protocol (see supplementary methods).Primary antibodies: rabbit anti-insulin receptor β , goat anti-actin . Secondary antibodies: rabbit anti-goat immunoglobulins/HRP , goat anti-rabbit immunoglobulins/HRP .Data presented consists of a comparison between the gray area in InsR-KO and InsR-Ctrl samples in ImageJ .2/40% O2, followed by decapitation. Perirenal, epididymal and gonadal white adipose tissues (WAT) were dissected upon sacrifice for evaluation of fat mass gain.Basal blood glucose levels of ad libitum fed animals were measured at the age of 7 weeks. Blood was obtained from a puncture in the tail vein. Glucose levels (mmol/L) were measured with a glucometer . Obesity was induced at the age of 8 weeks with a diet containing 58 kcal% fat and 25 kcal% carbohydrates , for the duration of 10 weeks. Control animals were fed a standard chow diet . Body weight (BW), and cumulative food and water intake were monitored twice per week throughout the whole study. Data presented in this manuscript is the BW gain per week and average food and water intake per animal per week. At 18 weeks of age, i.e., after 10 weeks of HFD exposure, respiratory exchange rate (RER), locomotor activity, and heat production were measured by a customized indirect gas calorimetric system . In short, chow- or HFD-fed mice were single housed in metabolic cages equipped with a PhenoMaster module for indirect gas calorimetry for 3 days for adaptation, followed by a continuous 48-h cycle of measurement of oxygen consumption (VO2) and carbon dioxide production (VCO2), as well as XT activity , and Z activity  4 times per hour. These parameters were used for the calculation of the RER, activity, and energy expenditure (EE). After 12 weeks of diet exposure, the animals were sacrificed by euthanasia with 60% COAn intraperitoneal insulin tolerance test (ipITT) was performed at 19 weeks of age by injecting an insulin bolus   in 4 h-fasted mice. Tail blood glucose levels (mmol/L) were measured with a glucometer  before (0 min) and at 20, 30, 45, 60, 90, and 120 min after injection.Blood plasma was used to evaluate fasted insulin concentration with the Rat/Mouse Insulin ELISA Kit , following the manufacturer’s instructions. The kit has inter-assay precision 6.0–17.9% and intra-assay precision 0.9–8.4%, and a lower detection limit 0.2 ng/mL. All samples were measured in duplicate.® 480 (Roche Life Science) for Hcrt, Agrp, Npy, Cd68, Il1b and Ikbkb genes . Gene expression is presented as a relative fold change to housekeeping genes (Hprt and bActin).Hypothalamic gene expression was evaluated using quantitative PCR (qPCR). Briefly, RNA was isolated using Tri-reagent  and the ISOLATE II RNA Mini Kit  following the manufacturer’s guidelines. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit  following the manufacturer’s guidelines. qPCR was performed with the LightCyclerAnimals were sacrificed at 20–21 weeks of age by perfusion fixation (see supplementary methods). Hypothalamic 30 µm thick coronal sections were selected from each mouse covering the rostral–caudal arcuate (ARC) nucleus region. Immunohistochemical staining of the ionized calcium-binding adaptor molecule 1 (Iba1)- and proopiomelanocortin (POMC)-ir were performed to profile, respectively, the microglial morphology and the neighboring POMC neurons; immunofluorescent co-staining of Iba1 and CD68 was performed to examine microglial phagocytic capacity in the ARC region. See supplementary methods for further information on staining protocols.Primary antibodies: rabbit anti-iba1 , rabbit anti-POMC , rat anti-CD68 .Secondary antibodies: goat anti-rabbit IgG biotinylated , goat anti-rat IgG, biotinylated . Fluorescent secondary antibodies: Streptavidin, Alexa Fluor 488 , goat anti-rabbit IgG, Alexa Fluor 594 .t-test. Data are presented as mean ± SEM. Results were considered statistically significant when p < 0.05.Statistical analyses were performed using Graphpad PRISM . Statistical significance was determined using one-way, two-way ANOVA and student


SARS-CoV-2 has caused the COVID-19 pandemic since early 2020. As of January 2022, the worldwide spreading of SARS-CoV-2 leads to approximately 0.35 billion of human infections and five millions of deaths. Current vaccination is one of the effective ways to control SARS-CoV-2 transmission and reduce the disease severity. However, the antibody level against the immunogen significantly drops several months after the standard two-dose vaccination, and hence a third or fourth dose booster (the same immunogen) has been suggested to boost the antibody response. Here, we described an ultra-effective nasal vaccine booster that potently induced the extraordinary high-level of neutralizing antibody in pre-vaccinated mice. The vaccine booster is composed of a recombinant receptor binding domain of SARS-CoV-2 spike (either wild-type or omicron) fused with a domain of SARS-CoV-2 nucleoprotein. In the absence of adjuvants, a single intranasal administration of the booster in pre-vaccinated mice significantly induced systemic and mucosal antibody responses as evidenced by the elevation of the cross-variant neutralizing antibody and induction of IgA in bronchoalveolar lavage respectively. Most importantly, the single dose nasal vaccine booster (omicron version) potently enhanced the neutralizing activity against authentic SARS-CoV-2 omicron virus infection. Taken together, the induction of respiratory mucosal immunity and the enhancement of cross-variant neutralizing activity by the nasal vaccine booster warrants further clinical trials in humans. As of 26 January 2022, the SARS-CoV-2 virus has caused for more than 350 million confirmed COVID-19 cases of infection worldwide. Vaccination remains the most crucial preventive measure against the disease. Currently, several types of COVID-19 vaccines have been widely employed, including inactivated whole virions, adenoviruses, protein subunits and mRNA vaccines. Two doses of intramuscular vaccination of either of the vaccines generally elicit high-level neutralizing antibodies (nAbs) that can effectively neutralize the original SARS-CoV-2 and the subsequently emerged variant strains , but to a lesser extent to delta variants , some ofWT), which composes of spike receptor-binding domain (RBD) fused with a domain of nucleocapsid protein, as a nasal vaccine booster without adjuvant. A mouse vaccination model was designed to determine the booster efficacy , implying that the nasal booster not only enhanced the preimmunized anti-spike RBD antibody response but also functioned as a new immunogen. Further study on the induction of lung resident memory B cells and follicular helper T cells by nasal booster will reveal the underlying mechanism on the induction of mucosal immunity after primary intramuscular vaccination.With respect to this, we designed and tested the novel idea of using a chimeric SARS-CoV-2 protein (N-RBDWT booster by omicron RBD could further increase the neutralization activity against the omicron variant without compromising the augmentation of neutralization against wild-type SARS-CoV-2 and delta variant. We repeated the immunization experiment in a mouse model and compared the neutralization activity induced by the booster of N-RBDWT and N-RBDOmicron. Serum and BAL fluid were obtained at 42 days post-immunization for authentic virus neutralization assay (illustrated in 50 = 6 × 103, black bar in 50 = 5 × 102, black bar in WT and N-RBDOmicron booster potently enhanced the neutralizing activity against wild-type, D614G, delta and omicron virus infection (orange and green bars in Omicron potently enhanced the neutralizing activity against authentic omicron virus infection (green bars in Omicron was unprecedently effective, reaching FRNT50 = 1.5×104. Since the omicron variant predominantly infects the upper respiratory tract and displays high transmissibility, further development of N-RBDOmicron into human nasal vaccine booster could help to control the spreading of the circulating variants including omicron.Several reports clearly demonstrated that antibodies induced by the two-dose intramuscular injection of spike mRNA vaccine are less neutralizing against omicron variant ,10. It iThe use of recombinant proteins for vaccination has previously been explored. By applying the protein vaccine intranasally, it is possible to induce mucosal immunity to combat infections of the mucosa theoretically. Nonetheless, recent studies demonstrated that nasal protein vaccines could induce adaptive immune response only in the presence of strong adjuvants such as Alum and STING-agonist , indicatClick here for additional data file.


To enable datasearching and downloading of these cis- and trans-eQTLs,we developed a user-friendly database named JAMIR-eQTL, publicly available at https://www.jamir-eqtl.org/. This isthe first miR-eQTL database designed for dementia types. Our integrative and comprehensiveresource will contribute to understanding the genetic basis of miRNA expression as well asto the discovery of deleterious mutations, particularly in dementia studies.MicroRNAs (miRNAs) are small non-coding RNAs shown to regulate gene expression by bindingto complementary transcripts. Genetic variants, including single-nucleotide polymorphismsand short insertions/deletions, contribute to traits and diseases by influencing miRNAexpression. However, the association between genetic variation and miRNA expressionremains to be elucidated. Here, by using genotype data and miRNA expression data from 3448Japanese serum samples, we developed a computational pipeline to systematically identifygenome-wide miRNA expression quantitative trait loci (miR-eQTLs). Not only did we identifya total of 2487 Database URL: https://www.jamir-eqtl.org/ MicroRNAs (miRNAs) are small non-coding RNA (ncRNA) molecules of ∼22 nucleotides that playkey roles in the post-transcriptional regulation of gene expression and control ofbiological processes in development, differentiation, growth, apoptosis and metabolism.miRNAs have been the focus of recent studies as potential biomarkers of a variety ofdiseases, including cancers , 2, cardThe development of high-throughput genotyping and massive parallel next-generationsequencing enables the genotyping of a large number of genetic variants, includingsingle-nucleotide polymorphisms (SNPs) and short insertions/deletions (indels). Genome-wideassociation studies have successfully identified numerous genetic variants associated withhuman complex traits , 11 and Expression quantitative trait locus (eQTL) analysis is a powerful approach to elucidatingthe biological correlations between gene expression and genetic variants and helps toexplain the regulatory mechanisms underlying trait- or disease-associated loci , 16. Altcis-eQTLs and trans-eQTLs. Ourintegrative and comprehensive resource will contribute to understanding the genetic basis ofmiRNA expression, as well as to the discovery of deleterious mutations associated with humancomplex diseases, including dementia. Furthermore, since miRNAs have been reported to beassociated with many diseases including cancer  Biobank were used and represented six types of dementia as well as ‘Other’, which included all samples notrepresenting one of the six dementia types. The numbers of subjects were 1314 for AD, 134for DLB, 69 for VaD, 31 for FTLD, 39 for NPH, 504 for MCI and 1357 for Other. All subjectsprovided written informed consent. The study was approved by the NCGG ethicalcommittee.Genotyping data were downloaded from the NCGG Biobank database. Those data were obtainedaccording to established protocols. Genomic DNA was extracted from peripheral bloodleukocytes by using standard protocols and a Maxwell RSC [Rapid Sample Concentrator]Instrument (Promega). All 3448 subjects were genotyped by using the Affymetrix Japonicagenotyping array  . Genotyphttp://www.mirbase.org/). miRNAexpression data were normalized by using the following four steps: (i) calculation of meanand standard deviation (SD) by using a set of pre-selected negative control signals from which the top and bottom 5% of values had been removed; (ii)replacement of signal values greater than the mean + 2 SD of the background signals withlog2 and labeling as effective signals; (iii) replacement of the remainingsignal values with the minimum of the effective signals−0.1 and (iv) replacement ofundetected signal values with the minimum signal of each miRNA. Finally, we used a set ofpre-selected internal control miRNAs , which we hadfound were stably expressed in more than 500 serum samples, to normalize the signalsacross different microarrays. Each miRNA signal value was standardized by using its ratioto the average signal of three internal control miRNA signals  designed todetect 2562 miRNA sequences registered in miRBase release 21 ..http://wcis-miR-eQTLs were defined as genetic variants located within 1 Mbupstream or downstream of the mature miRNA sequence, and trans-miR-eQTLswere defined as genetic variants located beyond 1 Mb upstream or downstream of the maturemiRNA sequence or on another chromosome. The genomic locations of mature miRNA sequenceswere retrieved from the miRBase database   . We examp.shtml) . Matrix subjects .cis- and trans-miR-eQTLs inthe six dementia types and all subjects. We identified a total of 2487cis-miR-eQTLs and 3 155 773 trans-miR-eQTLs at anFDR <0.05 in six dementia types and all samples, including those from patients withother types of dementia. We identified 542 cis- and 668 069trans-miR-eQTLs in AD, 1 cis- and 17 788trans-eQTLs in DLB, 96 cis- and 150 609trans-eQTLs in VaD, 4659 trans-eQTLs in FTLD, 2022trans-eQTLs in NPH, 1326 cis- and 1 694 332trans-eQTLs in MCI, and 522 cis- and 618 294trans-eQTLs in all subjects ,P-values and FDRs  format.When users search by using a genetic variant, miRNA or genomic location of interest, thedatabase returns a table of related miR-eQTL records, consisting of dementia types,and FDRs . Allcoland FDRs . A vectoClicking on the variant related to the miR-eQTL provides the minor allele, major alleleand minor allele frequency in our database . ClickinOnly one ncRNA-related eQTL database, namely ncRNA-eQTL , has recThe JAMIR-eQTL database was constructed by using Golang with a PostgreSQL backend-database.A Nuxt.js application was used to build the front-end interface.https://www.jamir-eqtl.org/.JAMIR-eQTL is freely available to all users without restriction at


Metallo-betalactamases (MBL) are rapidly becoming a more widespread form of antimicrobial resistance. MBL are class B betalactamases that use zinc rather than serine in their active site and are only inactivated by monobactams, such as aztreonam. Unfortunately, most MBL-producing organisms also produce aztreonam-inactivating beta-lactamases. Synergy between ceftazidime-avibactam and aztreonam is well documented for MBL-producing Enterobacteriaceae but has not been tested extensively in non-fermenting Gram-negative bacteria. This study evaluates the susceptibilities of non-fermenting Gram-negative bacteria via E-test to this combination in vitro, in order to provide support for use to treat infections from these organisms.Pseudomonas aeruginosa, Pseudomonas putida, and the intrinsically aztreonam resistant Acinetobacter baumanii using the E-test method. MBL-producing Enterobacteriaceae were included as positive controls. All isolates were also tested against ceftazidime alone, aztreonam alone, and ceftazidime-avibactam. Bacterial isolates were procured from the Multidrug-resistant organism Repository & Surveillance Network at the Walter Reed Army Institute of Research. Antimicrobial resistance genes were previously identified by whole genome sequencingThe antibiotic combination ceftazidime-avibactam+aztreonam was tested against a total of 33 isolates, including MBL-producing Pseudomonas spp. isolates tested, 9 were resistant, 3 were intermediate, and 1 was susceptible to aztreonam. Synergistic testing of ceftazidime-avibactam+aztreonam reduced the MIC of 4 Pseudomonas isolates by 1-2 doubling dilutions. While Acinetobacter spp. are usually considered intrinsically resistant to aztreonam, synergistic testing of ceftazidime-avibactam+aztreonam reduced the MIC of all 12 isolates tested by 1 to 3 doubling dilutions.Of 13 Acinetobacter baumanii and MBL-producing Pseudomonas aeruginosa is a potentially promising therapeutic option when faced with growing antimicrobial resistance.The ability of ceftazidime-avibactam+aztreonam to reduce the MICs of All Authors: No reported disclosures


COVID-19 vaccine-induced myocarditis is a rare adverse event in the current pandemic. The following is a case series of 10 individuals with COVID-19 vaccine-related myocarditis confirmed by cardiac magnetic resonance imaging. In this cohort of predominantly male patients, with a mean age of 23 years, chest discomfort and positive cardiac biomarkers occurred at a median of 3 days after the second COVID-19 vaccine dose. Although systolic function was relatively preserved on noninvasive cardiac imaging, evidence was seen of delayed enhancement on cardiac magnetic resonance imaging, confirming myocarditis. As COVID-19 vaccine-induced myocarditis has a relatively benign clinical course, the benefits of vaccination still, by far, outweigh this small risk. The COVID-19 pandemic has resulted in unprecedented morbidity and mortality, and it has had economic and social impacts that will continue for decades to come. In response to the pandemic, 2 mRNA vaccines were developed, by Pfizer and Moderna, respectively, and were approved by Health Canada in December 2020. Since that time, a small number of case reports have described the rare occurrence of myocarditis after COVID-19 vaccination, raising the confirmation of this rare vaccine-related adverse event. We report a case series of 10 patients diagnosed with COVID-19 vaccine-related myocarditis by cardiac magnetic resonance (CMR) imaging.All CMR studies positive for myocarditis between March and September 2021 at the St. Boniface Hospital in Winnipeg, Manitoba, Canada were evaluated. Patients were identified as having COVID-19 vaccine-induced myocarditis as adapted from the case definition and classification of the Brighton Collaboration (Pandemic Emergency Process).Between March 16, 2021 and September 3, 2021, out of 42 cases of CMR-confirmed myocarditis, a total of 10 individuals presented with COVID-19 vaccine-related myocarditis . The meaOn transthoracic echocardiography (TTE), the mean left ventricular ejection fraction (LVEF) was 56%, with mild regional wall motion abnormalities . All patients demonstrated a preserved or borderline reduced LVEF on CMR imaging . On CMR All patients were admitted to the hospital for work-up of myocarditis and monitoring, with a median stay of 4.5 days (range: 3-6 days). The majority of cases were treated with ibuprofen and colchicine for acute pericarditis, and although 2 patients required additional therapy for recurrent chest pain, no adverse events, including arrhythmias, heart failure, cardiogenic shock, and death, were noted in any of the 10 cases.COVID-19 vaccine-induced myocarditis is a rare adverse event in the current pandemic.A number of mechanisms have been proposed to explain the finding of myocardial inflammation following COVID-19 vaccination. First, molecular mimicry between the SARS-CoV-2 spike protein, encoded for by the mRNA vaccines, and cardiac molecules may elicit an immune response in predisposed individuals.,Similar to previous case series, our findings confirm that COVID-19 vaccine-related myocarditis disproportionately affects young men, primarily after the second vaccine dose.The echocardiographic and CMR findings of a normal or borderline reduced LVEF in our case series were consistent with previous reports.•Myocarditis is a rare side effect following COVID-19 vaccination, overwhelmingly affecting young men after the second mRNA vaccine dose.•The clinical courses of our 10 patients were benign.•All patients demonstrated LGE on CMR imaging, which most commonly affected the inferior, inferolateral, and lateral walls in a subepicardial or mid-myocardial distribution.•Abnormal epicardial global longitudinal strain by echocardiography may be an additional noninvasive tool to identify COVID-19 vaccine-related myocarditis.Our study describes 10 cases of myocarditis identified using CMR imaging following COVID-19 vaccination. In this case series, young men were more commonly affected, particularly after the second mRNA vaccine dose. As COVID-19 vaccine-induced myocarditis has a relatively benign clinical course, the benefits of vaccination still, by far, outweigh this small risk.


Accumulating evidence suggests that anti-CD20 treatments are associated with a more severe course of COVID-19. We present the case of a 72-year-old woman treated with the B-cell-depleting anti-CD20 antibody rituximab for seropositive rheumatoid arthritis with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing a clinical relapse more than 4 weeks after the first manifestation. Persistently positive reverse transcription polymerase chain reaction (RT-PCR) results along with a drop in cycling threshold (Ct) values, in addition to recovery of identical viral genotype by whole genome sequencing (WGS) during the disease course, argued against reinfection. No seroconversion was noted, as expected on anti-CD20 treatment. Several other case reports have highlighted potentially fatal courses of COVID-19 associated with B-cell-depleting treatments. Immunocompromised patients are at risk for severe disease courses of COVID-19 and prolonged viral shedding . Here, wth of February 2021. Her past medical history was remarkable for seropositive rheumatoid arthritis and connective tissue disease-associated interstitial lung disease, for which she received rituximab treatment biannually. Five months before COVID-19 onset, she had received her last dose. Other comorbidities included pulmonary arterial hypertension, coronary artery disease, and atrial fibrillation. At the early stage of the national vaccination campaign, our patient had not been vaccinated. Twelve days later, she was hospitalized in another hospital due to respiratory failure. In addition to the known interstitial lung disease, chest computed tomography revealed new bilateral ground glass opacities consistent with COVID-19 infiltrates. The patient received supplemental oxygen, dexamethasone, and a 10-day course of remdesivir. Following her gradual improvement, a nasopharyngeal SARS-CoV-2 antigen test turned out negative on day 22 and follow-up Ct values of RT-PCR were increasing; thus, isolation precautions were stopped. On day 27, the patient was discharged to inpatient pulmonary rehabilitation. Thirty-three days after diagnosis, she was admitted to our hospital because of relapsing dyspnea, productive cough, and fever, along with respiratory failure requiring nasal high-flow oxygen therapy and intensive care monitoring. Differential diagnoses included bacterial pneumonia, reinfection with a SARS-CoV-2 mutant, or a relapse of preexisting COVID-19. Inflammatory markers were again elevated, and a computed tomography scan was unchanged . Viral genotypes showed identical sequence patterns, and mutational analysis for N501Y and E484K was negative. Furthermore, SARS-CoV-2 serology was not able to detect IgG or IgM antibodies. These findings led to the conclusion that our patients' clinical deterioration was due to a prolonged and relapsing course of COVID-19.Our literature review showed accumulating evidence for variable clinical courses of COVID-19 in patients with functional B-cell immunodeficiency due to treatment with rituximab .Reported patients mostly suffered from hemato-oncological or rheumatological comorbidities as an indication for rituximab treatment. Prolonged shedding of SARS-CoV-2 in nasopharyngeal swabs along with either a benign or adverse outcome is a notable feature, independent of the underlying disease. To our knowledge, only one case report has provided whole genome sequencing to rule out reinfection during a prolonged clinical course , and reiOur single case description may not be generalizable to other immunocompromised populations. However, cases with relapsing and prolonged courses have been attributed to reduced viral clearance due to the lack of anti-SARS-CoV-2 antibody production by prolonged B-cell depletion after anti-CD20 therapy, as was the case in our patient , 10, 17.This case also highlights the infection control challenges in the handling of this special population with persistent shedding of potentially viable virus.In conclusion, caution should be taken in patients with anti-CD20 antibody treatment, as they can acquire SARS-CoV-2 infection despite vaccination because of the lack of antibody emergence and prolonged or relapsing disease courses have to be expected.


Extended community testing constitutes one of the main strategic pillars in controlling the COVID-19 pandemic. Reverse transcription PCR (RT-PCR) targeting the SARS-CoV-2 genome on nasopharyngeal swab samples is currently the reference test. While displaying excellent analytical sensitivity and specificity, this test is costly, often requires a substantial turnaround time, and, more importantly, is subject to reagent and other material shortages. To complement this technology, rapid antigen tests have been developed and made available worldwide, allowing cheap, quick, and decentralized SARS-CoV-2 testing. The main drawback of these tests is the reduced sensitivity when RT-PCR is the gold standard. In this study, we evaluate Visby an innovative, portable, easy-to-use RT-PCR point-of-care (POC) diagnostic device. Our retrospective analysis shows that overall, compared to the Cobas 6800 RT-qPCR assay (Roche), this RT-PCR POC technology detects SARS-CoV-2 RNA with 95% sensitivity (95%CI = 86.3–99%) and 100% specificity (95% CI = 80.5–100%). For samples with cycle-threshold values below 31, we observed 100% sensitivity (95% CI = 66.4–100%). While showing an analytical sensitivity slightly below that of a standard RT-qPCR system, the evaluated Visby RT-PCR POC device may prove to be an interesting diagnostic alternative in the COVID-19 pandemic, potentially combining the practical advantages of rapid antigen tests and the robust analytical performances of nucleic acid detection systems. Complementary to prevention measures, such as social distancing, mask wearing, and hand hygiene, extensive population screening with isolation of positive cases is one of the key means of controlling the COVID-19 pandemic caused by severe SARS-CoV-2 [Rapid antigen tests are increasingly widely used, allowing for user-friendly, low-cost, quick, and decentralized testing. The main limitation of these tests is the reduced sensitivity in comparison to RT-qPCR, around 90% in the best-case scenario. Innovative nucleic acid detection point-of-care (POC) diagnostic technologies are now being developed, potentially combining the excellent analytical performance of RT-qPCR and the practical advantages of rapid antigen tests ,4.An alternative to the detection of antigen is the amplification of nucleic acid by reverse transcription in combination with isothermal methods such as: loop-mediated isothermal amplification (RT-LAMP) ,6,7, rechttps://www.fda.gov/media/145917/download, accessed on 28 April 2021). Of note, the device may only be used once before requiring disposal or refurbishment by the manufacturer. Visby is intended for testing individuals suspected of COVID-19 by their health care provider using nasopharygeal, anterior nasal or mid-turbinate swabs collected into transport media,Recently, Visby Medical Inc. (Visby) has developed and begun manufacturing small palm-sized RT-PCR devices capable of detecting SARS-CoV-2. This device contains all necessary reagents, and is operated simply by loading a sample and sequentially pushing buttons; thus representing a self-contained easy-to-use RT-PCR POC diagnostic device. The device makes use of a continuous flow serpentine PCR channel combined with an enzyme linked detection chemistry to produce a colorimetric signal that is easily observable by the user. The device, originally developed for detection of bacterial targets in a sexually transmitted infection panel  has beenIn this study, we tested the new RT-PCR POC Device developed by Visby to detect the presence of SARS-CoV-2 RNA in a retrospective study using frozen SARS-CoV-2 positive nasopharyngeal samples using Cobas 6800 RT-qPCR assay (Roche) as the gold standard.TM or an in-house DMEM-based media that has been validated in our institution [According to the Swiss Ethics Committee on research involving leftover clinical samples, this study occurred in the context of a method quality validation in an emergency setting and therefore did not require any authorization from our ethics committee. The vast majority of positive samples come from outpatient symptomatic patients enrolled in a COVID-19 screening center at the Geneva University Hospitals. Sixty-one confirmed positive SARS-CoV-2 nasopharyngeal swab (NPS) samples were tested. Sample positivity, that is samples with a cycle-threshold (Ct) value below 35, was previously determined by using the Cobas 6800 SARS-CoV-2 RT-PCR assay  and stored in Copan UTMtitution . Sampleshttps://www.fda.gov/media/142228/download, accessed on 28 April 2021) with some modifications, as we performed a retrospective study using frozen SARS-CoV-2 samples. Briefly, specimens were mixed by inversion 5 times, and 650 µL was inserted into provided dilution buffer using the special Pasteur pipette provided with the device   A. The diMedical) B. SampleResults of valid devices were displayed in the test result window as visible purple marks B. A purpThe validated Roche Cobas 6800 SARS-CoV-2 RNA extraction and RT-qPCR assay were used as a reference assay . In case of discrepant results between the Cobas and Visby assays, viral RNA genome detection was also performed using GeneXpert (Ref: XPRSARS-CoV2-10 from Cepheid). Cobas targets ORF1a/b and a pan-Sarbecovirus conserved region of the E gene, while GeneXpert targets N and E genes.The sensitivity and specificity of the device were determined by comparison to the Cobas assay, according to sensitivity = TP/(TP + FN); Specificity = TN/(TN + FP); where TP = true-positives, FN = false-negatives, TN = true-negatives, and FP = false-positives. Confidence intervals of 95% (95% CI) were calculated using the Clopper–Pearson method . We repoTM, Epithelix SARL, Geneva, Switzerland) were washed three times with PBS  and incubated in MucilAirTM medium at 33 °C and 5% CO2. Four days post infection, respiratory tissues culture supernatant is removed and 200µL of MucilAirTM medium was added apically and viral culture supernatant was collected after 20 min of incubation at 33 °C at 5% CO2. Viral load in the culture supernatant was determined by RNA quantification. Briefly, RNA was extracted with NucliSens easyMAG , and quantified by RT-qPCR using SuperScript™ III Platinum™ One-Step qRT-PCR Kit  in a CFX96 Thermal Cycler . RT-qPCR was performed using a specific set of primers and probes targeting the SARS-CoV-2 E gene  [6 copies/mL.As recommended by the manufacturer, a SARS-CoV-2 negative nasopharyngeal cell matrix was prepared to perform LoD analyses. First, nasopharyngeal samples, which were previously verified to be SARS-CoV-2 negative, were pooled to obtain a total of 12 mL. The pooled nasopharyngeal sample was retested by Cobas RT-qPCR to ensure that the sample pool was negative and immediately frozen at −80 °C. SARS-CoV-2 viral stock was produced as follows: SARS-CoV-2 isolates were collected from ex vivo infections of airway epithelia cultured in an air-liquid interface as previously described . Briefly-BBQ-3′) . In vitrFrozen samples were thawed, dispensed immediately into dilution buffer provided with the device, and further processed to avoid nucleic acid damage as much as possible. After 30 min of sample processing, purple mark recording was performed immediately in devices displaying the green light. An invalid run occurred for a single sample, but after retesting, yielded a valid result.The clinical accuracy of the device compared to Cobas RT-qPCR was calculated. From the 61 Cobas-positive samples , the device detected 58 positive samples with 3 false-negative samples. All 17 Cobas-negative samples were also found to be negative by the device . We obseSince discrepant results might be explained by the effects of sample freezing on RNA stability, we retested the discordant samples with the Cobas assay. Nearly identical positive Cobas Ct values were found, showing that RNA degradation upon thawing samples could not explain the negative device results . Discrep6 copies/mL. Live virus from cell culture supernatant were serially diluted  in SARS-CoV-2 negative nasopharyngeal cell matrix to evaluate the LoD. We tested a single replicate of each dilution and found them all positive, showing a Visby positive detection with low viral loads (102 copies/mL). Our data are in agreement with the manufacturer LoD determination, which showed a 95% detection rate for 1112 genomic copies/mL and a detection rate of 45% for 125 genomic copies/mL .A clinical SARS-CoV-2 isolate was cultured in an air-liquid interface respiratory epithelium system (mucilAir). Viral supernatant concentration was quantified using E-gene standards from the European virus archive and diluted to a final concentration of 2.1 × 10The aim of this study was to evaluate the Visby POC device to detect the presence of SARS-CoV-2 RNA in previously collected frozen clinical nasopharyngeal samples, in comparison to the Cobas 6800 RT-qPCR assay (Roche). The devices detected SARS-CoV-2 RNA with 95% sensitivity and 100% specificity.https://www.fda.gov/media/142228/download, accessed on 28 April 2021). Having excluded potential issues related to different viral genes targeted with both assays, the reduced sensitivity observed with the devices probably results from the different technologies applied, which differ in technical details such as buffers and sample processing methods. Visby’s test is not directly compatible with most viral transport media (manufacturer package insert). While dilution of samples collected in transport media mitigates the inhibitory effects of the transport media, it reduces the test sensitivity, which could account for the 3 Cobas-positive samples that the device missed. However, this dilution step did not prevent detection of other samples with similar viral loads. We cannot exclude the possibility of PCR inhibiting substances within these nasal samples that prevent gene amplification exclusively with the device methodology [Few discrepant results were observed that might be explained by potential N-gene mutations that are undetectable by the device, similarly to previously published observations showing that failure to detect SARS-CoV-2 by the Cobas 6800 assay was linked to E-gene mutations . This isDilution of the sample collected into transport media to mitigate the inhibitory effects of the transport media may reduce the sensitivity of the test. While a remnant sample evaluation was performed in this study, it is reasonable to assume that direct swab elution with no dilution step would yield an improvement in sensitivity. A prospective clinical study would be important to assess the clinical sensitivity of the direct swab method.In the present analytical study, the POC Device performs less well at Ct values ≥35; however, the reduction in sensitivity is relatively unimportant since high Ct values probably indicate a low transmission risk . FurtherThis device operates by detecting viral nucleic acids such as the reference standard (RT-PCR) and isothermal amplification tests. RT-LAMP amplification unfortunately has issues such as: non-specific amplification that can be a problem in later steps as a result of “carry-over contamination”, “product cross contamination” ,29,30, oIn contrast, the device evaluated here uses the same robust and well established principle used by the gold-standard tests, and thus is expected to be reliable and accurate. Other RT-PCR devices suitable for POC diagnostics are on the market  but are less portable than the Visby POC device evaluated here, and may be unavailable or have a sample processing time of up to 90 min that may be prohibitive for some envisioned POC uses .It is important to note the limitations of our study. The selection of samples tested is not representative of an epidemiological trend because only frozen samples were used, and no prospective recruitment of patients was performed with both methods concomitantly. No estimation of positive or negative predictive values can be performed as samples were not consecutively collected and prevalence is not interpretable. Future clinical evaluations, including head-to-head comparisons with antigen tests and isothermal tests, testing of SARS-CoV-2 asymptomatic-contacts, and screening healthcare workers will help to better position SARS-CoV-2 nucleic acid detection among the POC diagnostics.In summary, the Visby device appears to be an attractive alternative POC test, performing nearly as well as conventional RT-qPCR tests, while displaying the appealing characteristics of a POC test. It is available in a portable format with minimal requirement of technical skills. After sample acquisition, less than 1 min is needed for treatment and loading samples onto de VISBY device. Viral extraction, amplification and easy-readout are obtained after exactly 30 min. This short turnaround time allows for rapid patient isolation or orientation decisions, e.g., in nursing homes or even hospital settings lacking more complex laboratory equipment. A potential disadvantage includes the generation of waste that remains an issue to be addressed. The company is exploring recycling components to reduce generated waste.


An analogous paradigm has been recently developed in cancer immunotherapy by the generation of CAR T-cells in vivo. These new ideas on endogenous reprogramming, cross-fertilized from the fields of regenerative medicine and gene therapy, are opening exciting avenues for direct modulation of immune or tumor cells in situ, widening our strategies to remove cancer immunotherapy roadblocks. Here, we review current strategies for cancer immunotherapy, summarize technologies for generation of immune cells by cell fate reprogramming as well as highlight the future potential of inducing these unique cell identities in vivo, providing new and exciting tools for the fast-paced field of cancer immunotherapy.Advances in understanding how cancer cells interact with the immune system allowed the development of immunotherapeutic strategies, harnessing patients’ immune system to fight cancer. Dendritic cell-based vaccines are being explored to reactivate anti-tumor adaptive immunity. Immune checkpoint inhibitors and chimeric antigen receptor T-cells (CAR T) were however the main approaches that catapulted the therapeutic success of immunotherapy. Despite their success across a broad range of human cancers, many challenges remain for basic understanding and clinical progress as only a minority of patients benefit from immunotherapy. In addition, cellular immunotherapies face important limitations imposed by the availability and quality of immune cells isolated from donors. Cell fate reprogramming is offering interesting alternatives to meet these challenges. Induced pluripotent stem cell (iPSC) technology not only enables studying immune cell specification but also serves as a platform for the differentiation of a myriad of clinically useful immune cells including T-cells, NK cells, or monocytes at scale. Moreover, the utilization of iPSCs allows introduction of genetic modifications and generation of T/NK cells with enhanced anti-tumor properties. Immune cells, such as macrophages and dendritic cells, can also be generated by direct cellular reprogramming employing lineage-specific master regulators bypassing the pluripotent stage. Thus, the cellular reprogramming toolbox is now providing the means to address the potential of patient-tailored immune cell types for cancer immunotherapy. In parallel, development of viral vectors for gene delivery has opened the door for  Cancer progression entails close crosstalk between tumor cells and the immune system. The evolution of cancer is driven by oncogenic mutations and the deregulation of important signaling pathways. The unleashed proliferative capacity leads to the accumulation of additional mutations, endowing tumor cells with selective advantages such as improved fitness and survival. Yet, such cancer diversity resulting from high mutational burden comes with a cost. The more mutations cancer cells acquire, the more likely they are recognized and eliminated by the immune system due to the expression of various tumor-associated antigens (TAAs) –3. Howevvia exposure to bacterial toxins  are engineered to selectively infect and replicate in tumor cells and to induce immunogenic death. Hypothetically, the administration of OV therapy should be able to target natural cancer-immunity interactions at multiple levels: OV-mediated lysis of tumor cells promotes the release of TAAs and type I interferon, boosting anti-tumor immunity.Since the first report of conditionally replicating Herpes Simplex type 1 (HSV-1) engineered virus which prolonged survival in nude mice with glioma , a myriaFurthermore, the engineering of OVs allows enforced expression of genes with therapeutic potential, including TAAs, costimulatory molecules, and cytokines , which can further amplify immune responses and counteract tumor-induced immunosuppression. The only FDA-approved oncolytic viral-based therapy is talimogene laherparepvec (T-VEC), used in advanced melanoma patients Figure 1Given their unique characteristics, OVs are promising therapeutic agents, but their efficacy, when used as a single therapy, leaves room for improvement. In this way, the versatility of the oncolytic viral platform makes it suitable for combination with other immunotherapies and conventional treatments. The combination of T-VEC with immune checkpoint inhibitors showed improved efficacy in melanoma without an increase in adverse effects, delivering long-term response in up to 62% of patients –41. The Immune checkpoints were originally discovered as a group of evolutionarily conserved molecules acting as negative regulators of T-cell activation, with cytotoxic T-lymphocyte Antigen 4 (CTLA-4) and PD-1 inhibitors being recently approved for clinical practice. CTLA-4 is primarily found on T-cells and controls early-stage T-cell activation by counteracting the activity of the co-stimulatory molecule CD28 –45. UnliInhibition of immune checkpoint receptors employs synthesized antibodies as a therapeutic intervention disrupting the ability of some cancer cells to evade a patient´s immune response . AdminisDespite delivering unprecedented long-lasting anti-tumor responses , persistFollowing the therapeutic success in metastatic melanoma and NSCLC, checkpoint inhibitors became increasingly used as a treatment alternative in a range of solid tumors, particularly in cancers attributed to carcinogen exposure or infectious origin. In addition to NSCLC, smoking is associated with the development of bladder cancer, head and neck carcinoma, renal cell carcinoma, or stomach malignancies, conferring a high tumor mutational burden –60. ImpoHarnessing T-cells for immunotherapy has long been of prime interest to cancer research. T-cells are unique immune cells: they are long-lived in resting conditions and proliferate extensively when encountering specific stimuli. T-cells elicit specific, robust, and enduring responses against tumor cells. As their action is systemic, they can reduce the tumor burden at the primary site and eradicate distant metastatic sites. On the downside, the manufacturing of lymphocytes for immunotherapy is challenging as it is so far tailored for each individual patient.ex vivo, and reinfused into patients, TILs induce rapid tumor regression  (First attempts to use T-cells in immunotherapy were based on tumor-infiltrating lymphocytes (TILs), a pool of T-lymphocytes pre-trained to recognize patient´s specific cancer cells. When expanded, pre-activated gression , 69. In val 93%) . DespiteChimeric antigen receptors (CAR) T-cells represent another breakthrough in immunotherapy. CAR T-cells are genetically engineered to express a synthetic T-cell receptor tailored to recognize surface antigens on cancer cells. The current generations of chimeric receptors usually consist of a single-chain variable fragment portion of the target receptor, along with the costimulatory intracellular domain of CD28, 4-1BB, or OX40 . CD19 taDespite achieving tremendous efficiency in the treatment of hematological malignancies, clinical success with CAR T-cells in the context of solid tumors is less encouraging, having hit multiple roadblocks from a limited number of targetable antigens to the inefficient trafficking of CAR T-cells into the tumor sites. Nevertheless, attempts are being made with CAR T-cells armed against ERBB2/HER2 receptor tyrosine kinase frequently over-expressed by tumor cells, as well as targeting TAAs including mesothelin, carcinoembryonic antigen (CEA), GD2 –79, or Ivia the direct interaction of native NK receptors with ligands on target cells. They are specially competent in recognizing cells that have lost MHC-I expression  patients from leukemia relapse , which lvia directed differentiation of induced Pluripotent Stem Cells (iPSCs), paving the way to harness cell reprogramming for immunotherapy . However, clinical exploitation of iPSC technology has several major challenges.Reprogramming to pluripotency was successfully shown from multiple somatic cell populations including fibroblasts, keratinocytes, neural progenitors, and peripheral blood cells –102, demin vitro. In addition, the iPSC differentiation process is very dependent on line-to-line variability, and generates a heterogeneous population of cells, hampering clinical translation. Importantly, despite epigenetic resetting during induction of pluripotency, iPSCs may retain residual parental epigenetic memory which may bias their differentiation potential into certain cell types based on the source of donor cells used to establish iPSC , are pluripotent and retain the capacity to generate all cell types of the embryo when provided with stage-specific signaling allowing differentiation to the cell type of interest. It is however a major challenge to fully recapitulate this process ish iPSC . There iantibody , while aantibody .via endothelial-to-hematopoietic transition in several waves, in vitro differentiation of hematopoietic precursors then requires multiple steps involving the formation of embryonic bodies allowing mesodermal development. Furthermore, a range of differentiation cytokine cocktails, as well as co-culture with feeder layers promote cell growth and differentiation  can contribute to tumor clearance by the production of IL-12 and other pro-inflammatory cytokines, generation of reactive oxygen species, and presentation of antigens from engulfed dead tumor cells. However, cues produced by tumor cells can polarize macrophages into M2 anti-inflammatory phenotype, inducing an immunosuppressive microenvironment and supporting tumor development .Several publications reported successful differentiation of macrophages from iPSCs upon culture with IL-3 and M-CSF , 127. iPin vitro and in a xenograft murine model  specific to β-amyloid to help devour amyloid plaques, bringing new potential for the treatment of Alzheimer’s disease . A similne model . Macrophne model .via combined expression of the transcription factor CEBPα or CEBPβ, which favors myeloid development by the downregulation of B-cell commitment transcriptional factor PAX5, and PU.1 which is critical for instructing myeloid transcriptional program , and IL-4 (enriching in cDC1 and cDC2-like cells), as well as the maturation signals TNFα and LPS .Sachamitr et al. reported the directed differentiation of iPSCs into CD141+ cDC1 cells resembling naturally occurring cDC1s. Consistently with their capacity for antigen presentation, CD141+ iPSC-DCs acquired immunostimulatory CD40+ CD86+ phenotype upon TLR engagement and could elicit activation and proliferation of naïve T-cells. However, induced DCs produced only limited amounts of IL-12 cytokine and in a steady state displayed tolerogenic features characteristic for tissue-resident DCs . CD141+ via antigen cross-presentation  . The iPScificity , 160. Incificity , 163. Inphocytes .in vivo conversion of pro-pre-B cells into functional early T-cell lineage progenitors by transient expression of HOXB5. Although the conversion occurred in the bone marrow, early T-cell lineage progenitors migrate to the thymus and mature into functional polyclonal T-cells , breast carcinoma cells were diverted into post-mitotic functional adipocytes with a cocktail composed of TGF-β, BMP2, insulin, and dexametazone. Notably, rosiglitazone-induced EMT together with adipogenic differentiation therapy efficiently prevented cancer cell invasion and formation of metastasis in vivo . Howeverotential .A large body of evidence indicates that inhibition of antigen presentation is one of the major mechanisms underlying cancer immune evasion, allowing tumor cells to become invisible to immune effector cells. Therefore, restoration or enforcement of antigen presentation machinery in cancer cells holds enormous potential to reactivate cytotoxic anti-tumor responses. While all cell types can present their own antigens through HLA class I molecules, only professional antigen-presenting cells, such as DCs or macrophages, can prime productive T-cell responses by providing costimulatory signals besides HLA class I/II and TCR interactions. Interestingly, expression of class I and class II molecules in tumor cells can be, at least to a certain extent, induced by radiotherapy, several chemotherapeutic agents, a range of small compounds including histone deacetylase (HDAC) inhibitors, tyrosine kinase inhibitors , CDK4/6 in vitro generated cells. In parallel with ex vivo based therapy, the development of viral vectors for gene delivery has opened opportunities for in vivo reprogramming in regenerative medicine, an elegant strategy circumventing current limitations of in vitro cell manipulation. Therefore, the possibility of switching cell identity within the living organism gained attention for its immense therapeutic potential. The main advantages of inducing immune cell reprogramming in vivo would be the use of internal cell sources, limited immune rejection, limited risk for tumor formation and increased reprogramming efficiency and fidelity , highlighting a possible convergence of approaches towards cancer immunotherapy . Their targeted elimination esponses . Howeveresponses , also imesponses .The immunogenicity of cancer cells is another fundamental determinant of the immune response. The strategy to increase tumor immunogenicity reported by Wang et al. consisted of intratumoral delivery of activating CRISPRa components to augment endogenous expression of TAAs. In mouse melanoma and pancreatic adenocarcinoma models, CRISPRa-mediated enhanced expression of TAAs increased the proportion of antigen-presenting cells and T-lymphocytes within the tumor microenvironment and elicited systemic anti-tumor immunity. Notably, the developed platform can be modified to direct simultaneous activation of expression of multiple TAAs as well as other immunomodulatory molecules, chemokines, or cytokines to further support cancer immune responses . A stratA riveting solution to boost tumor immunogenicity and to fuel local anti-cancer immune responses was recently suggested by Nissim et al. who engineered lentiviral-delivered synthetic gene circuits driving expression of immune-modulating components including a surface T-cell engager, the chemokine ligand CCL21, IL-12, and anti-PD1 antibody directly in tumor cells. To prevent unwanted toxicity and the risk of autoimmunity due to the off-target delivery into neighboring non-malignant cells, the expression of immune-modulating agents was linked to the activation of cancer-specific promoters, ensuring that the immune response is directed exclusively against cancer cells. Interestingly, the proposed immune-modulating therapy elicited a robust T-cell mediated response and resulted in the rejection of ovarian cancer .Ex vivo produced CAR T-cells recognizing tumor antigens has proved as a highly effective treatment for patients with refractory B-cell malignancies. The manufacturing process which requires extensive and time-consuming procedures is the limiting step. To simplify the process, Pfeiffer et al. recently developed lentiviral vectors enabling highly selective targeting of CD8+ cells which is used for in vivo generation of CAR T-cells. Despite the relatively low transduction efficiency (not exceeding 5%), in vivo engineered CD19-CAR T-cells demonstrated the ability to eliminate CD19+ cells in mice , the Swedish research council (2020-00615), European Research Council (866448-TrojanDC), Novo Nordisk Foundation (0056527) and FCT (PTDC/MED-IMU/4520/2020). The Knut and Alice Wallenberg foundation, the Medical Faculty at Lund University and Region Skåne are acknowledged for generous financial support. OZ is supported by a Cancerfonden postdoctoral fellowship 190008. IC and AGF are supported by FCT doctoral fellowships (PD/BD/146424/2019 and SFRH/BD/133233/2017).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CASP9 polymorphisms have been linked with various cancers, neurological disorders, autoimmune pathologies and lumbar disc disease. Clinical reports suggest alterations in caspase-9 expression, activity or function may be associated with acute and chronic neurodegeneration, retinal neuropathy, slow-channel myasthenic syndrome, lumbar disc disease, cardiomyopathies, atherosclerosis and autoimmune disease. Healthy tissues maintain caspase-9 activity at low basal levels, rendering supraphysiological caspase-9 activation a tractable target for therapeutic interventions. Strategies for selective inhibition of caspase-9 include dominant negative caspase-9 mutants and pharmacological inhibitors derived from the XIAP protein, whose Bir3 domain is an endogenous highly selective caspase-9 inhibitor. However, the mechanistic implications of caspase-9 expression and activation remain indeterminate in many pathologies. By assembling clinical reports of caspase-9 genetics, signaling and cellular localization in human tissues, this review identifies gaps between experimental and clinical studies on caspase-9, and presents opportunities for further investigations to examine the consequences of caspase activity in human disease.Caspase-9, a cysteine-aspartic protease known for its role as an initiator of intrinsic apoptosis, regulates physiological cell death and pathological tissue degeneration. Its nonapoptotic functions, including regulation of cellular differentiation/maturation, innate immunity, mitochondrial homeostasis, and autophagy, reveal a multimodal landscape of caspase-9 functions in health and disease. Recent work has demonstrated that caspase-9 can drive neurovascular injury through nonapoptotic endothelial cell dysfunction.  Caspases are a conserved family of cysteine-aspartic proteases that mediate programmed cell death and inflammation. Cells express caspases as inactive zymogens, which activate rapidly in response to cellular signals. Initiator caspases  activate through dimerization upon binding to an activating platform. Once active, initiator caspases activate downstream effector caspases  by proteolytic cleavage, which can in turn activate additional initiator caspases . Consequin vitro and in vivo experimental studies with clinical literature on caspase-9 involvement in human disease.Here, we focus our attention on clinical reports of altered caspase-9 activity or function in human tissues to provide an overview of the clinical correlates between caspase-9 and disease pathologies. Due to the lack of clinicals approaches for modulating caspase-9 activity, these studies are by necessity correlative. However, functional human genetics and animal models provide mechanistic insight into the role of caspase-9 in disease progression. Our goal is to assemble a framework linking Caspase-9 activation occurs in the apoptosome, a heptameric structure comprised of Apaf-1 (Apoptotic protease activating factor 1) and cytochrome c, which forms in response to permeabilization of the mitochondrial outer membrane and release of cytochrome c into the cytosol  Bratton. Bindingvia direct cleavage of vimentin  is a clinical-stage technology developed by Bellicum, which involves pre-transplant transfection of donor T-cells with a chemically inducible caspase-9 gene that can be activated by a small molecule dimerization enhancer , portal hypertension, and liver failure/cirrhosis. The treatment was generally well tolerated, supporting the clinical feasibility of targeting caspases as a therapeutic strategy, but failed to meet primary efficacy endpoints . The lacCASP9 (as well as in apoptosis-related genes CASP3 and APAF1) have been identified in cases of neural tube defects (NTDs)  . Liu et s (NTDs)  charactes (NTDs) . The H23tability . Meanwhitability . ProteinCASP9 deletion in murine studies, which results in embryonic lethality due to severe brain malformations and hindbrain neural tube defects  receptor) in B cells and ICOS (inducible T-cell costimulator) in T cells.Caspase-9 coding variants have also been associated with immune system pathologies. Localized deletion of murine caspase-9 from hematopoietic progenitors impairs leukocyte proliferation and differentiation , and dysCASP9 polymorphisms have also been associated with autoimmune and hematopoietic pathologies. The coding variant A28V/G (rs1052571) is associated with lower risk of severe ulcerative colitis (CASP9 expression across multiple tissues by eQTL (expression Quantitative Trait Loci) analysis on the GTex Portal. While it has been speculated that the Q221R variant may cause a conformational change modifying the affinity of caspase-9 to Apaf-1 , a neuromuscular disease driven by degeneration of motor neurons in the brain and spinal cord. Inoue et al.  identifiThe association of increased caspase-9 expression with neurodegeneration is not limited to chronic neurodegenerative diseases. Two studies reported increased levels of full-length and cleaved caspase-9 in CSF of patients with traumatic brain injury (TBI) . AlthougNeurodegenerative pathologies features upregulated caspase-9 in neuronal and glial cells, as well as in patient CSF and blood serum samples. These studies indicate that caspase-9 mediates common pathways of neuronal injury across multiple neurodegenerative pathologies, and suggest increasing caspase-9 involvement in later stages of disease when neuronal death is prevalent. However, further study is needed to elucidate how different neurodegenerative etiologies induce caspase-9 activation, to clarify the significance of caspase-9 activity in non-neuronal cell types, and to determine whether targeting caspase-9 activity can offer a therapeutic strategy for treating neurodegenerative disease. Given the multimodal role of caspase-9 in regulating neuronal circuit organization, mitochondrial homeostasis, innate immune response, and neurovascular barrier disruption, it is highly likely that caspase-9 involvement in neurodegenerative pathologies is likewise heterogeneous.Retinal cell death contributes to permanent vision loss in ophthalmic disease, and neuroprotective treatment strategies may translate to vision-saving treatments for retinal disorders such as photoreceptor degeneration, diabetic retinopathy, and glaucoma . Several+ photoreceptors in four retinas from donors with retinal detachment. However, it is not clear if degenerating photoreceptors are the primary source of cleaved caspase-9 in subretinal fluid from RRD. Intriguingly, it is endothelial caspase-9 activity that regulates severity of retinal detachment and subsequent photoreceptor injury in mouse studies  and in intervertebral disk degeneration in lumbar disc disease (LDD) .Vohra et al.  reportedA combination of genetic and histologic studies have identified an association between caspase-9 and lumbar disc disease (LDD). Lumbar disk disease occurs when intervertebral disks degenerate, bulge from the bony area of the lower spine, and eventually herniate. The herniated disk can press on nerve roots in the lumbar spine, causing back pain, weakness or numbness. Treatment strategies for LDD depend on severity of disease, ranging from conservative pain management to surgical interventions that excise herniated lumbar segments, and restorative pharmacological treatments are still in early experimental stages of development .CASP9 polymorphisms in patients with lower back pain and identified a significant association with the rs4645978 polymorphism in the CASP9 promoter region. This association has since been replicated in two other studies  lesions compared to unaffected intima, initial lesions, lipofibrous plaques or fibrous plaques from thoracic aorta biopsies. Additionally, the A28V/G mutation in caspase-9 has been associated with decreased risk of ischemic stroke  renders the heart globally ischemic, and can trigger myocardial injury upon reperfusion. Feng et al.  showed iTung et al.  examinedCaspase-9 involvement has also been reported in nonischemic myocardial pathologies. Three studies have implicated caspase-9 in dilated cardiomyopathy (DCM), a form of myocardial degeneration that causes weakening and enlargement of the heart. Aharinejad et al.  reportedTo date, studies of caspase-9 involvement in heart disease have relied primarily on biochemical detection methods, and we still do not know the cellular localization of caspase-9 in these pathologies, limiting our understanding of the signaling pathways regulating caspase-9 in cardiomyopathy. These reports remain agnostic regarding whether caspase signaling mediates cell death in DCM patients. On the other hand, CP/Rep and chronic rejection of heart transplant clearly link caspase-9 activation with cell death. Correlational studies indicate a close association between caspase-9 expression and disease severity, however further interventional studies in animal models of DCM would be helpful to establish whether inhibiting caspase-9 may provide therapeutic benefit in these disorders.Caspase-9 has multiple apoptotic and nonapoptotic cellular functions, which complicates interpretations of caspase-9 expression in disease tissues. While colocalization of caspase-9 with dying neurons has been established in several neurodegenerative conditions, its nonapoptotic roles in immune regulation, endothelial function, mitochondrial homeostasis and neuronal circuit organization substantially expand the potential scope of caspase-9 involvement in disease . In partCASP9 SNPs are specifically associated with alterations in caspase-9 signaling. Furthermore, coding variants A28V/G (rs1052571) and Q221R (rs1052576) are still not characterized for how they may impact caspase-9 signaling, despite extensive associations of these mutants with multiple cancers and degenerative and inflammatory pathologies. Understanding the functional consequence of these mutations would provide key genetic evidence to determine if caspase-9 is a disease-causing target in diseases associated with these variants.Human genetic evidence implicates caspase-9 loss of function mutations with neural tube defects, pediatric tumors, and immunodeficiency/lymphoproliferation. These loss of function phenotypes help elucidate the diverse roles played by caspase-9 in different tissues, but are not readily tractable for caspase-9-targeted therapeutic interventions due to the inherent risks of induced cell death caused by caspase-9 upregulation. Gene association studies suggest potential caspase-9 involvement with multiple cancers, autoimmune disorders, and neurological disease. Clinicogenomic analysis of caspase-9 activity and expression in patient tissues should help clarify IF putative pathogenic Pathogenic expression and activation of caspase-9 has been reported in neuronal, vascular, lumbar, myocardial and hematopoietic tissues. Interventional studies in animal models support caspase-9 as a driver of disease pathology in ALS, retinal detachment, and SCS, suggesting that inhibiting caspase-9 activity can be a viable therapeutic strategy for these disorders. However, for the majority of human pathologies associated with upregulated caspase-9, the therapeutic potential of caspase-9 inhibition strategies has not yet been tested. Exploring caspase signaling across different organ systems can help expand our understanding of degenerative diseases by examining cell-type specific dysfunction subsequent to caspase-9 activation. Eye research is readily translatable to CNS disease due to the shared neurovascular architecture of brain and retinal tissues , and cas