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BACKGROUND: SARS-CoV-2 infection during pregnancy is associated with an increased risk for stillbirth, preeclampsia, and preterm birth. However, this does not seem to be caused by intrauterine fetal infection because vertical transmission is rarely reported. There is a paucity of data regarding the associated placental SARS-CoV-2 histopathology and their relationship with the timing and severity of infection. OBJECTIVE: This study aimed to determine if maternal SARS-CoV-2 infection was associated with specific patterns of placental injury and if these findings differed by gestational age at time of infection or disease severity. STUDY DESIGN: A retrospective cohort study was performed at the University of California San Diego between March 2020 and February 2021. Placentas from pregnancies with a positive SARS-CoV-2 test were matched with 2 sets of controls; 1 set was time-matched by delivery date and sent to pathology for routine clinical indications, and the other was chosen from a cohort of placentas previously collected for research purposes without clinical indications for pathologic examination before the SARS-CoV-2 outbreak. Placental pathologic lesions were defined based on standard criteria and included maternal and fetal vascular malperfusion and acute and chronic inflammatory lesions. A bivariate analysis was performed using the independent Student t test and Pearson chi-square test. A logistic regression was used to control for relevant covariates. Regions of SARS-CoV-2-associated villitis were further investigated using protein-based digital spatial profiling assays on the GeoMx platform, validated by immunohistochemistry, and compared with cases of infectious villitis and villitis of unknown etiology. Differential expression analysis was performed to identify protein expression differences between these groups of villitis. RESULTS: We included 272 SARS-CoV-2 positive cases, 272 time-matched controls, and 272 historic controls. The mean age of SARS-CoV-2 affected subjects was 30.1±5.5 years and the majority were Hispanic (53.7%) and parous (65.7%). SARS-CoV-2 placentas demonstrated a higher frequency of the 4 major patterns of placental injury (all P<.001) than the historic controls. SARS-CoV-2 placentas also showed a higher frequency of chronic villitis and severe chronic villitis (P=.03 for both) than the time-matched controls, which remained significant after controlling for gestational age at delivery (adjusted odds ratio, 1.52; 95% confidence interval, 1.01-2.28; adjusted odds ratio, 2.12; 95% confidence interval, 1.16-3.88, respectively). Digital spatial profiling revealed that programmed death-ligand 1 was increased in villitis-positive regions of the SARS-CoV-2 (logFC, 0.47; adjusted P value =.002) and villitis of unknown etiology (logFC, 0.58; adjusted P value =.003) cases, but it was conversely decreased in villitis-positive regions of the infectious villitis group (log FC, -1.40; adjusted P value <.001). CONCLUSION: Chronic villitis seems to be the most specific histopathologic finding associated with SARS-CoV-2 maternal infection. Chronic villitis involves damage to the vasculosyncytial membrane of the chorionic villi, which are involved in gas and nutrient exchange, suggesting potential mechanisms of placental (and perhaps neonatal) injury, even in the absence of vertical transmission. Surprisingly, changes in protein expression in SARS-CoV-2-associated villitis seem to be more similar to villitis of unknown etiology than to infectious villitis.
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BACKGROUND: Cervical spinal (c-spine) injuries range greatly in severity from minor ligamentous injuries to osteoligamentous instability with spinal cord injuries. Initial evaluation begins with stabilization as needed and immediate immobilization. Current practice as to whether the c-spine can be cleared clinically without radiographic evaluation is often guided by using the National Emergency X-Radiography Utilization Study Low-Risk Criteria and the Canadian C-Spine Rule. Under these clinical decision guidelines, stable trauma patients presenting with alcohol intoxication cannot have the c-spine cleared clinically and imaging should be "considered." OBJECTIVE: This study aimed to assess the frequency of computed tomography (CT) c-spine scans ordered for patients presenting with alcohol intoxication to the emergency department (ED), the timing of the studies, and subsequently determine the proportion of which showed a clinically significant result that required intervention. METHODS: In this retrospective medical record review, all clinically alcohol-intoxicated patients presenting to two academic EDs were included. Overall demographic characteristics, time to order of CT imaging, radiology reads, and outcomes of patient visits were determined. RESULTS: There were 8008 patient visits included in the study. Of these visits, 5 patients scanned in ≤3 h had acute findings on CT scan and no patients with a deferred timing of CT scan after patients metabolized had an acute finding on CT scan. No patients required operative management. CONCLUSIONS: This study's results suggest that it is a safe clinical practice to defer CT imaging for patients presenting to the ED with alcohol intoxication and low suspicion for c-spine injury per history and examination.
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Intoxicação Alcoólica , Traumatismos da Coluna Vertebral , Ferimentos não Penetrantes , Humanos , Estudos Retrospectivos , Canadá , Tomografia Computadorizada por Raios X/métodos , Vértebras Cervicais/lesões , Serviço Hospitalar de Emergência , Traumatismos da Coluna Vertebral/diagnósticoRESUMO
Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical with those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.
Assuntos
Hidrolases Anidrido Ácido/genética , Glicólise/genética , Metaboloma/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Polifosfatos/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Linhagem Celular Transformada , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hidrólise , Metabolômica/métodos , Mitocôndrias/genética , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransgenesRESUMO
Alzheimer's Disease (AD) is the most common neurodegenerative disorder in our society, as the population ages, its incidence is expected to increase in the coming decades. The etiopathology of this disease still remains largely unclear, probably because of the highly complex and multifactorial nature of AD. However, the presence of mitochondrial dysfunction has been broadly described in AD neurons and other cellular populations within the brain, in a wide variety of models and organisms, including post-mortem humans. Mitochondria are complex organelles that play a crucial role in a wide range of cellular processes, including bioenergetics. In fact, in mammals, including humans, the main source of cellular ATP is the oxidative phosphorylation (OXPHOS), a process that occurs in the mitochondrial electron transfer chain (ETC). The last enzyme of the ETC, and therefore the ulterior generator of ATP, is the ATP synthase. Interestingly, in mammalian cells, the ATP synthase can also degrade ATP under certain conditions (ATPase), which further illustrates the crucial role of this enzyme in the regulation of cellular bioenergetics and metabolism. In this collaborative review, we aim to summarize the knowledge of the presence of dysregulated ATP synthase, and of other components of mammalian mitochondrial bioenergetics, as an early event in AD. This dysregulation can act as a trigger of the dysfunction of the organelle, which is a clear component in the etiopathology of AD. Consequently, the pharmacological modulation of the ATP synthase could be a potential strategy to prevent mitochondrial dysfunction in AD.
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Doença de Alzheimer/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/enzimologia , Animais , Metabolismo Energético , Humanos , Fosforilação OxidativaRESUMO
Notch signaling is an evolutionarily conserved pathway associated with the development and differentiation of all metazoans. It is needed for proper germ layer formation and segmentation of the embryo and controls the timing and duration of differentiation events in a dynamic manner. Perturbations of Notch signaling result in blockades of developmental cascades, developmental anomalies, and cancers. An in-depth understanding of Notch signaling is thus required to comprehend the basis of development and cancer, and can be further exploited to understand and direct the outcomes of targeted cellular differentiation into desired cell types and complex tissues from pluripotent or adult stem and progenitor cells. In this chapter, we briefly summarize the molecular, evolutionary, and developmental basis of Notch signaling. We will focus on understanding the basics of Notch signaling and its signaling control mechanisms, its developmental outcomes and perturbations leading to developmental defects, as well as have a brief look at mutations of the Notch signaling pathway causing human hereditary disorders or cancers.
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Desenvolvimento Embrionário , Neoplasias/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Humanos , Neoplasias/patologia , Receptores Notch/genética , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Inherited vascular malformations are commonly autosomal dominantly inherited with high, but incomplete, penetrance; they often present as multiple lesions. We hypothesized that Knudson's two-hit model could explain this multifocality and partial penetrance. We performed a systematic analysis of inherited glomuvenous malformations (GVMs) by using multiple approaches, including a sensitive allele-specific pairwise SNP-chip method. Overall, we identified 16 somatic mutations, most of which were not intragenic but were cases of acquired uniparental isodisomy (aUPID) involving chromosome 1p. The breakpoint of each aUPID is located in an A- and T-rich, high-DNA-flexibility region (1p13.1-1p12). This region corresponds to a possible new fragile site. Occurrences of these mutations render the inherited glomulin variant in 1p22.1 homozygous in the affected tissues without loss of genetic material. This finding demonstrates that a double hit is needed to trigger formation of a GVM. It also suggests that somatic UPID, only detectable by sensitive pairwise analysis in heterogeneous tissues, might be a common phenomenon in human cells. Thus, aUPID might play a role in the pathogenesis of various nonmalignant disorders and might explain local impaired function and/or clinical variability. Furthermore, these data suggest that pairwise analysis of blood and tissue, even on heterogeneous tissue, can be used for localizing double-hit mutations in disease-causing genes.
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Tumor Glômico/genética , Paraganglioma Extrassuprarrenal/genética , Dissomia Uniparental/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Quebra Cromossômica , Cromossomos Humanos Par 1/genética , DNA/genética , Feminino , Tumor Glômico/patologia , Humanos , Hibridização in Situ Fluorescente , Masculino , Mutação/genética , Paraganglioma Extrassuprarrenal/patologia , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Programs that control early lineage fate decisions and transitions from embryonic to adult human cell types during development are poorly understood. Using human pluripotent stem cells (hPSCs), in the present study, we reveal reduction of Hedgehog (Hh) signaling correlates to developmental progression of hematopoiesis throughout human ontogeny. Both chemical- and gene-targetingmediated inactivation of Hh signaling augmented hematopoietic fate and initiated transitions from embryonic to adult hematopoiesis, as measured by globin regulation in hPSCs. Inhibition of the Hh pathway resulted in truncation of Gli3 to its repressor, Gli3R, and was shown to be necessary and sufficient for initiating this transition. Our results reveal an unprecedented role for Hh signaling in the regulation of adult hematopoietic specification, thereby demonstrating the ability to modulate the default embryonic programs of hPSCs.
Assuntos
Proteínas Hedgehog/genética , Hematopoese/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Adulto , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/fisiologia , Células Sanguíneas/metabolismo , Células Sanguíneas/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Análise em Microsséries , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transcriptoma , Proteína Gli3 com Dedos de ZincoRESUMO
Notch signaling regulates several cellular processes including cell fate decisions and proliferation in both invertebrates and mice. However, comparatively less is known about the role of Notch during early human development. Here, we examined the function of Notch signaling during hematopoietic lineage specification from human pluripotent stem cells of both embryonic and adult fibroblast origin. Using immobilized Notch ligands and small interfering RNA to Notch receptors we have demonstrated that Notch1, but not Notch2, activation induced hairy and enhancer of split 1 (HES1) expression and generation of committed hematopoietic progenitors. Using gain- and loss-of-function approaches, this was shown to be attributed to Notch-signaling regulation through HES1, which dictated cell fate decisions from bipotent precursors either to the endothelial or hematopoietic lineages at the clonal level. Our study reveals a previously unappreciated role for the Notch pathway during early human hematopoiesis, whereby Notch signaling via HES1 represents a toggle switch of hematopoietic vs endothelial fate specification.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Embrionárias/citologia , Endotélio Vascular/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Receptor Notch1/metabolismo , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores/metabolismo , Western Blotting , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Derme/citologia , Derme/metabolismo , Células-Tronco Embrionárias/metabolismo , Endotélio Vascular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Humanos , Técnicas Imunoenzimáticas , Células-Tronco Pluripotentes Induzidas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição HES-1RESUMO
PURPOSE: Retinoblastoma is the most common intraocular malignancy in children. Although new chemotherapeutic approaches have improved ocular salvage rates, novel therapies are required for patients with refractory intraocular and metastatic disease. Chimeric antigen receptor (CAR) T cells targeting glypican-2 (GPC2) are a potential new therapeutic strategy. EXPERIMENTAL DESIGN: GPC2 expression and its regulation by the E2F1 transcription factor were studied in retinoblastoma patient samples and cellular models. In vitro, we performed functional studies comparing GPC2 CAR T cells with different costimulatory domains (4-1BB and CD28). In vivo, the efficacy of local and systemic administration of GPC2 CAR T cells was evaluated in intraocular and leptomeningeal human retinoblastoma xenograft models. RESULTS: Retinoblastoma tumors, but not healthy retinal tissues, expressed cell surface GPC2, and this tumor-specific expression was driven by E2F1. GPC2-directed CARs with 4-1BB costimulation (GPC2.BBz) were superior to CARs with CD28 stimulatory domains (GPC2.28z), efficiently inducing retinoblastoma cell cytotoxicity and enhancing T-cell proliferation and polyfunctionality. In vivo, GPC2.BBz CARs had enhanced persistence, which led to significant tumor regression compared with either control CD19 or GPC2.28z CARs. In intraocular models, GPC2.BBz CAR T cells efficiently trafficked to tumor-bearing eyes after intravitreal or systemic infusions, significantly prolonging ocular survival. In central nervous system (CNS) retinoblastoma models, intraventricular or systemically administered GPC2.BBz CAR T cells were activated in retinoblastoma-involved CNS tissues, resulting in robust tumor regression with substantially extended overall mouse survival. CONCLUSIONS: GPC2-directed CAR T cells are effective against intraocular and CNS metastatic retinoblastomas.
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Glipicanas , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Retinoblastoma , Linfócitos T , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Retinoblastoma/imunologia , Retinoblastoma/patologia , Retinoblastoma/terapia , Camundongos , Receptores de Antígenos Quiméricos/imunologia , Glipicanas/imunologia , Glipicanas/antagonistas & inibidores , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/terapia , Neoplasias do Sistema Nervoso Central/imunologia , Neoplasias do Sistema Nervoso Central/secundário , Neoplasias do Sistema Nervoso Central/patologia , Modelos Animais de Doenças , FemininoRESUMO
Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.
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Gangliosídeos , Imunidade Inata , Imunoterapia Adotiva , Células Matadoras Naturais , Neuroblastoma , Receptores de Antígenos Quiméricos , Linfócitos T , Neuroblastoma/imunologia , Neuroblastoma/terapia , Neuroblastoma/patologia , Animais , Humanos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Camundongos , Gangliosídeos/imunologia , Linhagem Celular Tumoral , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Glipicanas/imunologia , Glipicanas/metabolismo , Microambiente Tumoral/imunologia , FemininoRESUMO
The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133(+) cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133(+) cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology.
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Ensaio de Unidades Formadoras de Colônias/métodos , Células Endoteliais/citologia , Células-Tronco/citologia , Antígeno AC133 , Adulto , Animais , Antígenos CD/análise , Diferenciação Celular , Células Cultivadas , Glicoproteínas/análise , Células-Tronco Hematopoéticas/citologia , Humanos , Receptores de Lipopolissacarídeos/análise , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/análise , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
INTRODUCTION: The coronavirus 2019 (COVID-19) pandemic has presented various unprecedented challenges to healthcare systems globally, prompting society to adopt new preventative strategies to curb spread of the disease. Those experiencing homelessness have been particularly impacted because of barriers to practicing social distancing, inability to isolate, and poor access to care. Project Roomkey was established in California as a statewide measure to provide non-congregate shelter options for individuals experiencing homelessness to properly quarantine. On goal in this study was to analyze the effectiveness of hotel rooms as a safe disposition alternative to hospital admission for patients experiencing homelessness and who were also positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: This was a retrospective, observational study that included chart review of patients who were discharged to the hotel from March 2020-December 2021. We recorded demographic information, index visit details, number of emergency department (ED) visits both a month prior to and following the index visit, admission rates, and number of deaths. RESULTS: During this 21-month study period, a total of 2,015 patients who identified as undomiciled were tested for SARS-COV-2 in the ED for various reasons. Of those patients, 83 were discharged from the ED to the hotel. Of the 83 patients, 40 (48.2%) ultimately tested positive for SARS-CoV-2 during their index visit. Two patients returned to the ED within seven days with COVID-19-related symptoms, and 10 patients within 30 days. Two patients required subsequent admission with COVID-19 pneumonia. No deaths were recorded within the 30-day follow-up period. CONCLUSION: The availability of a hotel served as a safe alternative to hospital admission for patients experiencing homelessness and who were either suspected or confirmed to have COVID-19. It is reasonable to consider similar measures in the management of other transmissible diseases for patients experiencing homelessness who require isolation.
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COVID-19 , SARS-CoV-2 , Humanos , Teste para COVID-19 , Hospitalização , HospitaisRESUMO
BACKGROUND: Antibody-drug conjugates (ADCs) that deliver cytotoxic drugs to tumor cells have emerged as an effective and safe anticancer therapy. ADCs may induce immunogenic cell death (ICD) to promote additional endogenous antitumor immune responses. Here, we characterized the immunomodulatory properties of D3-GPC2-PBD, a pyrrolobenzodiazepine (PBD) dimer-bearing ADC that targets glypican 2 (GPC2), a cell surface oncoprotein highly differentially expressed in neuroblastoma. METHODS: ADC-mediated induction of ICD was studied in GPC2-expressing murine neuroblastomas in vitro and in vivo. ADC reprogramming of the neuroblastoma tumor microenvironment was profiled by RNA sequencing, cytokine arrays, cytometry by time of flight and flow cytometry. ADC efficacy was tested in combination with macrophage-driven immunoregulators in neuroblastoma syngeneic allografts and human patient-derived xenografts. RESULTS: The D3-GPC2-PBD ADC induced biomarkers of ICD, including neuroblastoma cell membrane translocation of calreticulin and heat shock proteins (HSP70/90) and release of high-mobility group box 1 and ATP. Vaccination of immunocompetent mice with ADC-treated murine neuroblastoma cells promoted T cell-mediated immune responses that protected animals against tumor rechallenge. ADC treatment also reprogrammed the tumor immune microenvironment to a proinflammatory state in these syngeneic neuroblastoma models, with increased tumor trafficking of activated macrophages and T cells. In turn, macrophage or T-cell inhibition impaired ADC efficacy in vivo, which was alternatively enhanced by both CD40 agonist and CD47 antagonist antibodies. In human neuroblastomas, the D3-GPC2-PBD ADC also induced ICD and promoted tumor phagocytosis by macrophages, which was further enhanced when blocking CD47 signaling in vitro and in vivo. CONCLUSIONS: We elucidated the immunoregulatory properties of a GPC2-targeted ADC and showed robust efficacy of combination immunotherapies in diverse neuroblastoma preclinical models.
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Imunoconjugados , Neuroblastoma , Humanos , Camundongos , Animais , Glipicanas , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Antígeno CD47 , Neuroblastoma/tratamento farmacológico , Macrófagos , Microambiente TumoralRESUMO
Neuroblastoma evolution, heterogeneity, and resistance remain inadequately defined, suggesting a role for circulating tumor DNA (ctDNA) sequencing. To define the utility of ctDNA profiling in neuroblastoma, 167 blood samples from 48 high-risk patients were evaluated for ctDNA using comprehensive genomic profiling. At least one pathogenic genomic alteration was identified in 56% of samples and 73% of evaluable patients, including clinically actionable ALK and RAS-MAPK pathway variants. Fifteen patients received ALK inhibition (ALKi), and ctDNA data revealed dynamic genomic evolution under ALKi therapeutic pressure. Serial ctDNA profiling detected disease evolution in 15 of 16 patients with a recurrently identified variant-in some cases confirming disease progression prior to standard surveillance methods. Finally, ctDNA-defined ERRFI1 loss-of-function variants were validated in neuroblastoma cellular models, with the mutant proteins exhibiting loss of wild-type ERRFI1's tumor-suppressive functions. Taken together, ctDNA is prevalent in children with high-risk neuroblastoma and should be followed throughout neuroblastoma treatment. SIGNIFICANCE: ctDNA is prevalent in children with neuroblastoma. Serial ctDNA profiling in patients with neuroblastoma improves the detection of potentially clinically actionable and functionally relevant variants in cancer driver genes and delineates dynamic tumor evolution and disease progression beyond that of standard tumor sequencing and clinical surveillance practices. See related commentary by Deubzer et al., p. 2727. This article is highlighted in the In This Issue feature, p. 2711.
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DNA Tumoral Circulante , Neuroblastoma , Criança , Humanos , DNA Tumoral Circulante/genética , Mutação , Genômica/métodos , Neuroblastoma/genética , Progressão da Doença , Receptores Proteína Tirosina Quinases/genética , Biomarcadores Tumorais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
OBJECTIVE: Loss-of-function mutations in genes coding for transforming growth factor-beta/bone morphogenetic protein receptors and changes in nitric oxide(*) (NO(*)) bioavailability are associated with hereditary hemorrhagic telangiectasia and some forms of pulmonary arterial hypertension. How these abnormalities lead to seemingly disparate pulmonary pathologies remains unknown. Endoglin (Eng), a transforming growth factor-beta coreceptor, is mutated in hereditary hemorrhagic telangiectasia and involved in regulating endothelial NO(*) synthase (eNOS)-derived NO(*) production and oxidative stress. Because some patients with pulmonary arterial hypertension harbor ENG mutations leading to haplo insufficiency, we investigated the pulmonary vasculature of Eng(+/-) mice and the potential contribution of abnormal eNOS activation to pulmonary arterial hypertension. METHODS AND RESULTS: Hemodynamic, histological, and biochemical assessments and x-ray micro-CT imaging of adult Eng(+/-) mice indicated signs of pulmonary arterial hypertension including increased right ventricular systolic pressure, degeneration of the distal pulmonary vasculature, and muscularization of small arteries. These findings were absent in 3-week-old Eng(+/-) mice and were attributable to constitutively uncoupled eNOS activity in the pulmonary circulation, as evidenced by reduced eNOS/heat shock protein 90 association and increased eNOS-derived superoxide ((*)O(2)(-)) production in a BH(4)-independent manner. These changes render eNOS unresponsive to regulation by transforming growth factor-beta/bone morphogenetic protein and underlie the signs of pulmonary arterial hypertension that were prevented by Tempol. CONCLUSIONS: Adult Eng(+/-) mice acquire signs of pulmonary arterial hypertension that are attributable to uncoupled eNOS activity and increased (*)O(2)(-) production, which can be prevented by antioxidant treatment. Eng links transforming growth factor/bone morphogenetic protein receptors to the eNOS activation complex, and its reduction in the pulmonary vasculature leads to increased oxidative stress and pulmonary arterial hypertension.
Assuntos
Endotélio Vascular/fisiopatologia , Hipertensão Pulmonar/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Estresse Oxidativo/fisiologia , Telangiectasia Hemorrágica Hereditária/fisiopatologia , Animais , Antioxidantes/uso terapêutico , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Óxidos N-Cíclicos/uso terapêutico , Modelos Animais de Doenças , Endoglina , Endotélio Vascular/metabolismo , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Óxido Nítrico Sintase Tipo III/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Marcadores de Spin , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
How alpha and beta globin genes are organized and expressed in amniotes is of interest to researchers in a wide variety of fields. Data regarding this from avian species have been scarce. Using genomic and proteomic approaches, we present here our analysis of alpha and beta globins of zebra finch, a passerine bird. We show that finch alpha globin gene cluster has three genes (alphas 1-3), each orthologous to its chicken counterpart. Finch beta globin gene cluster has three genes (betas 1-3), with an additional pseudogene at the 3' end. Finch beta3 is orthologous to chicken betaA, but the orthology of beta1 and beta2 to chicken counterparts is less clear. All six finch globins are confirmed to encode functional proteins. Gene expression in both globin gene clusters is regulated developmentally. Adult finch blood has a globin profile similar to that of adult chicken, with high levels of beta3 and alpha3 and moderate levels of alpha2. Finch embryonic primitive blood exhibits a globin profile very different from that of equivalent stage chick embryos, with all six globins expressed at high levels. Overall, our data provide a valuable resource for future studies in avian globin gene evolution and globin switching during erythropoietic development.
Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Tentilhões/genética , Regulação da Expressão Gênica no Desenvolvimento , alfa-Globinas/genética , Globinas beta/genética , Envelhecimento/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Embrião não Mamífero/metabolismo , Eritropoese , Tentilhões/crescimento & desenvolvimento , Genoma , Dados de Sequência Molecular , Família Multigênica , Filogenia , Alinhamento de Sequência , alfa-Globinas/química , alfa-Globinas/metabolismo , Globinas beta/química , Globinas beta/metabolismoRESUMO
BACKGROUND: In addition to erythrocytes, embryonic blood contains other differentiated cell lineages and potential progenitor or stem cells homed to changing niches as the embryo develops. Using chicken as a model system, we have isolated an enriched pool of circulating non red blood cells (nRBCs) from E4 and E6 embryos; a transition period when definitive hematopoietic lineages are being specified in the peri-aortic region. RESULTS: Transcriptome analysis of both nRBC and RBC enriched populations was performed using chicken Affymetrix gene expression arrays. Comparison of transcript profiles of these two populations, with verification by RT-PCR, reveals in nRBCs an expression signature indicative of hematopoietic stem cells (HSCs) and progenitor cells of myeloid and lymphoid lineages, as well as a number of previously undescribed genes possibly involved in progenitor and stem cell maintenance. CONCLUSION: This data indicates that early circulating embryonic blood contains a full array of hematopoietic progenitors and stem cells. Future studies on their heterogeneity and differentiation potentials may provide a useful alternative to ES cells and perinatal blood.
Assuntos
Células Sanguíneas/citologia , Diferenciação Celular , Linhagem da Célula , Embrião de Galinha/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Animais , Separação Celular , Citometria de Fluxo , Perfilação da Expressão Gênica , Marcadores Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Clinically relevant and reliable reports derived from in vitro research are dependent on the choice of cell isolation protocols adopted between different laboratories. Peripheral blood eosinophils are conventionally isolated using density-gradient centrifugation followed by immunomagnetic selection (positive/negative) while neutrophils follow a more simplified dextran-sedimentation methodology. With the increasing sophistication of molecular techniques, methods are now available that promise protocols with reduced user-manipulations, improved efficiency, and better yield without compromising the purity of enriched cell populations. These recent techniques utilize immunomagnetic particles with multiple specificities against differential cell surface markers to negatively select non-target cells from whole blood, greatly reducing the cost/time taken to isolate granulocytes. Herein, we compare the yield efficiencies, purity and baseline activation states of eosinophils/neutrophils isolated using one of these newer protocols that use immunomagnetic beads (MACSxpress isolation) vs. the standard isolation procedures. The study shows that the MACSxpress method consistently allowed higher yields per mL of peripheral blood compared to conventional methods (P<0.001, n=8, Wilcoxon paired test), with high isolation purities for both eosinophils (95.0±1.7%) and neutrophils (94.2±10.1%) assessed by two methods: Wright's staining and flow cytometry. In addition, enumeration of CD63+ (marker for eosinophil activation) and CD66b+ (marker for neutrophil activation) cells within freshly isolated granulocytes, respectively, confirmed that conventional protocols using density-gradient centrifugation caused cellular activation of the granulocytes at baseline compared to the MACSxpress method. In conclusion, MACSxpress isolation kits were found to be superior to conventional techniques for consistent purifications of eosinophils and neutrophils that were suitable for activation assays involving degranulation markers.
Assuntos
Eosinófilos/fisiologia , Separação Imunomagnética/métodos , Neutrófilos/fisiologia , Centrifugação com Gradiente de Concentração , Citometria de Fluxo , Humanos , Contagem de LeucócitosRESUMO
The acquisition of innate immune response is requisite to having bona fide differentiation of airway epithelium. Procedures developed to differentiate lung airway from human pluripotent stem cells (hPSCs) have demonstrated anecdotal evidence for innate immune response, but an in-depth exploration of response levels is lacking. Herein, using an established method of airway epithelial generation from hPSCs, we show that hPSC-derived epithelial cells are able to up-regulate expression of TNFα, IL8 and IL1ß in response to challenge with bacterial endotoxin LPS, but lack response from genes associated with innate immune response in other cell types. Further, stimulation of cells with TNF-α resulted in auto-induction of TNFα transcript, as well as cytokine responses of IL8 and IL1ß. The demonstration of innate immune induction in hPSC-derived airway epithelia gives further strength to the functionality of in vitro protocols aimed at generating differentiated airway cells that can potentially be used in a translational setting. Finally, we propose that innate immune challenge of airway epithelium from human pluripotent stem cell sources be used as a robust validation of functional in vitro differentiation.