RESUMEN
Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.
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Melanoma , Linfocitos T , Ratones , Animales , Linfocitos T/patología , Neutrófilos/patología , Deriva y Cambio Antigénico , Inmunoterapia , Antígeno CTLA-4RESUMEN
There is an urgent need to create novel models using human disease-relevant cells to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology and to facilitate drug screening. Here, as SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs (particularly alveolar type-II-like cells) are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines following SARS-CoV-2 infection, similar to what is seen in patients with COVID-19. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high-throughput screen of drugs approved by the FDA (US Food and Drug Administration) and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid and quinacrine dihydrochloride. Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics.
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Antivirales/farmacología , COVID-19/virología , Colon/citología , Evaluación Preclínica de Medicamentos/métodos , Pulmón/citología , Organoides/efectos de los fármacos , Organoides/virología , SARS-CoV-2/efectos de los fármacos , Animales , COVID-19/prevención & control , Colon/efectos de los fármacos , Colon/virología , Aprobación de Drogas , Femenino , Xenoinjertos/efectos de los fármacos , Humanos , Técnicas In Vitro , Pulmón/efectos de los fármacos , Pulmón/virología , Masculino , Ratones , Organoides/citología , Organoides/metabolismo , SARS-CoV-2/genética , Estados Unidos , United States Food and Drug Administration , Tropismo Viral , Internalización del Virus/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Group 2 innate lymphoid cells (ILC2s) regulate inflammation and immunity in mammalian tissues1,2. Although ILC2s are found in cancers of these tissues3, their roles in cancer immunity and immunotherapy are unclear. Here we show that ILC2s infiltrate pancreatic ductal adenocarcinomas (PDACs) to activate tissue-specific tumour immunity. Interleukin-33 (IL33) activates tumour ILC2s (TILC2s) and CD8+ T cells in orthotopic pancreatic tumours but not heterotopic skin tumours in mice to restrict pancreas-specific tumour growth. Resting and activated TILC2s express the inhibitory checkpoint receptor PD-1. Antibody-mediated PD-1 blockade relieves ILC2 cell-intrinsic PD-1 inhibition to expand TILC2s, augment anti-tumour immunity, and enhance tumour control, identifying activated TILC2s as targets of anti-PD-1 immunotherapy. Finally, both PD-1+ TILC2s and PD-1+ T cells are present in most human PDACs. Our results identify ILC2s as anti-cancer immune cells for PDAC immunotherapy. More broadly, ILC2s emerge as tissue-specific enhancers of cancer immunity that amplify the efficacy of anti-PD-1 immunotherapy. As ILC2s and T cells co-exist in human cancers and share stimulatory and inhibitory pathways, immunotherapeutic strategies to collectively target anti-cancer ILC2s and T cells may be broadly applicable.
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Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/inmunología , Linfocitos/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Células Dendríticas/inmunología , Femenino , Humanos , Inmunidad Innata/inmunología , Inmunoterapia , Interleucina-33/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunologíaRESUMEN
Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.
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Vasos Sanguíneos/citología , Carcinogénesis , Células Endoteliales/citología , Hemodinámica , Neoplasias/irrigación sanguínea , Organogénesis , Organoides/irrigación sanguínea , Vasos Sanguíneos/crecimiento & desarrollo , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Cromatina/metabolismo , Epigénesis Genética , Epigenómica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Islotes Pancreáticos/irrigación sanguínea , Modelos Biológicos , Especificidad de Órganos , RNA-Seq , Análisis de la Célula Individual , Factores de Transcripción , TranscriptomaRESUMEN
BACKGROUND: Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive. METHODS: We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of Abcg2-expressing ECs to vessel development and regeneration, we developed Abcg2CreErt2;ROSA TdTomato mice and performed lineage tracing during mouse development and during tissue regeneration after myocardial infarction injury. RNA sequencing and chromatin methylation chromatin immunoprecipitation followed by sequencing were conducted to study the gene regulation in Abcg2-expressing ECs. RESULTS: In human and mouse vessels, ECs with higher ABCG2 expression (ABCECs) possess higher clonal proliferative potential and in vivo vessel-forming potential compared with mature ECs. These cells could clonally contribute to vessel formation in primary and secondary recipients after transplantation. These features of ABCECs meet the criteria of CRECs. Results from lineage tracing experiments confirm that Abcg2-expressing CRECs (AbcCRECs) contribute to arteries, veins, and capillaries in cardiac tissue development and vascular tissue regeneration after myocardial infarction. Transcriptome and epigenetic analyses reveal that a gene expression signature involved in angiogenesis and vessel development is enriched in AbcCRECs. In addition, various angiogenic genes, such as Notch2 and Hey2, are bivalently modified by trimethylation at the 4th and 27th lysine residue of histone H3 (H3K4me3 and H3K27me3) in AbcCRECs. CONCLUSIONS: These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Humanos , Ratones , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/citología , Neovascularización Fisiológica , Proliferación Celular , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/genética , Infarto del Miocardio/terapia , Regeneración , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ratones Transgénicos , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/citología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Linaje de la CélulaRESUMEN
BACKGROUND: Recent studies have demonstrated that individuals hospitalized due to COVID-19 can be affected by "long-COVID" symptoms for as long as one year after discharge. OBJECTIVES: Our study objective is to identify data-driven clusters of patients using a novel, unsupervised machine learning technique. METHODS: The study uses data from 437 patients hospitalized in New York City between March 3rd and May 15th of 2020. The data used was abstracted from medical records and collected from a follow-up survey for up to one-year post-hospitalization. Hospitalization data included demographics, comorbidities, and in-hospital complications. The survey collected long-COVID symptoms, and information on general health, social isolation, and loneliness. To perform the analysis, we created a graph by projecting the data onto eight principal components (PCs) and running the K-nearest neighbors algorithm. We then used Louvain's algorithm to partition this graph into non-overlapping clusters. RESULTS: The cluster analysis produced four clusters with distinct health and social connectivity patterns. The first cluster (n = 141) consisted of patients with both long-COVID neurological symptoms (74%) and social isolation/loneliness. The second cluster (n = 137) consisted of healthy patients who were also more socially connected and not lonely. The third cluster (n = 96) contained patients with neurological symptoms who were socially connected but lonely, and the fourth cluster (n = 63) consisted entirely of patients who had traumatic COVID hospitalization, were intubated, suffered symptoms, but were socially connected and experienced recovery. CONCLUSION: The cluster analysis identified social isolation and loneliness as important features associated with long-COVID symptoms and recovery after hospitalization. It also confirms that social isolation and loneliness, though connected, are not necessarily the same. Physicians need to be aware of how social characteristics relate to long-COVID and patient's ability to cope with the resulting symptoms.
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COVID-19 , Hospitalización , Soledad , Aislamiento Social , Humanos , COVID-19/epidemiología , COVID-19/psicología , Ciudad de Nueva York/epidemiología , Masculino , Femenino , Hospitalización/estadística & datos numéricos , Persona de Mediana Edad , Análisis por Conglomerados , Aislamiento Social/psicología , Anciano , Soledad/psicología , Adulto , Síndrome Post Agudo de COVID-19 , Aprendizaje Automático no Supervisado , SARS-CoV-2RESUMEN
Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.
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Endotelio Vascular/citología , Células Madre Pluripotentes/citología , Factores de Transcripción/genética , Animales , Barrera Hematoencefálica , Encéfalo/irrigación sanguínea , Encéfalo/citología , Diferenciación Celular , Línea Celular , Reprogramación Celular/fisiología , Endotelio Vascular/fisiología , Expresión Génica , Humanos , Ratones Endogámicos , Células Madre Pluripotentes/fisiología , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismoRESUMEN
Transplant-associated thrombotic microangiopathy (TA-TMA) is an endotheliopathy complicating up to 30% of allogeneic hematopoietic stem cell transplants (alloHSCT). Positive feedback loops among complement, pro-inflammatory, pro-apoptotic, and coagulation cascade likely assume dominant roles at different disease stages. We hypothesized that mannose-binding lectin-associated serine protease 2 (MASP2), principal activator of the lectin complement system, is involved in the microvascular endothelial cell (MVEC) injury characteristic of TA-TMA through pathways that are susceptible to suppression by anti-MASP2 monoclonal antibody narsoplimab. Pre-treatment plasmas from 8 of 9 TA-TMA patients achieving a complete TMA response in a narsoplimab clinical trial activated caspase 8, the initial step in apoptotic injury, in human MVEC. This was reduced to control levels following narsoplimab treatment in 7 of the 8 subjects. Plasmas from 8 individuals in an observational TA-TMA study, but not 8 alloHSCT subjects without TMA, similarly activated caspase 8, which was blocked in vitro by narsoplimab. mRNA sequencing of MVEC exposed to TA-TMA or control plasmas with and without narsoplimab suggested potential mechanisms of action. The top 40 narsoplimab-affected transcripts included upregulation of SerpinB2, which blocks apoptosis by inactivating procaspase 3; CHAC1, which inhibits apoptosis in association with mitigation of oxidative stress responses; and pro-angiogenesis proteins TM4SF18, ASPM, and ESM1. Narsoplimab also suppressed transcripts encoding pro-apoptotic and pro-inflammatory proteins ZNF521, IL1R1, Fibulin-5, aggrecan, SLC14A1, and LOX1, and TMEM204, which disrupts vascular integrity. Our data suggest benefits to narsoplimab use in high-risk TA-TMA and provide a potential mechanistic basis for the clinical efficacy of narsoplimab in this disorder.
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Anticuerpos Monoclonales Humanizados , Trasplante de Células Madre Hematopoyéticas , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Microangiopatías Trombóticas , Humanos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Caspasa 8/genética , Caspasa 8/uso terapéutico , Proteínas del Sistema Complemento , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidores , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Microangiopatías Trombóticas/tratamiento farmacológico , Microangiopatías Trombóticas/etiología , Microangiopatías Trombóticas/genética , Resultado del TratamientoRESUMEN
[Figure: see text].
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COVID-19/inmunología , Inmunidad Celular/inmunología , Mediadores de Inflamación/inmunología , Macrófagos/inmunología , Miocitos Cardíacos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/patología , Técnicas de Cocultivo/métodos , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Macrófagos/patología , Masculino , Persona de Mediana Edad , Miocardio/inmunología , Miocardio/patología , Miocitos Cardíacos/patología , Células Madre Pluripotentes/inmunología , Análisis de Secuencia de ARN/métodosRESUMEN
Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1+ FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFß and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.
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Diferenciación Celular , Reprogramación Celular , Endotelio/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Inmunidad Adaptativa , Envejecimiento/genética , Animales , Línea Celular , Linaje de la Célula , Autorrenovación de las Células , Células Clonales/citología , Células Clonales/trasplante , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
INTRODUCTION: Suicidality among young people is a significant societal issue. The current study conducted a meta-analysis of community and clinical interventions targeting suicide attempts, self-harm, and suicidal ideation in adolescents. METHODS: Interventions targeting suicide attempts, self-harm and suicidal ideation were identified by searching PsychINFO, Medline, CINAHL and Embase in line with the PRISMA statement. Study quality was determined using a risk of bias tool. Meta-analyses examined the efficacy of the interventions. Effect sizes were calculated for suicidal ideation data (continuous data) using Hedge's g for standardised mean differences. Suicide attempts and self-harm (dichotomous data) were calculated using odds ratios (ORs). RESULTS: Seventeen RCTs were included in the meta-analysis. No significant differences were found between treatment and control groups on measures of suicide attempts or self-harm. A small effect-size was observed on measures of suicidal ideation (g = 0.47). A secondary meta-analysis investigated change over time in treatment as usual conditions, finding significant large effect-sizes for suicide attempts (OR = 18.67), self-harm (OR = 12.77), and suicidal ideation (g = 0.86). LIMITATIONS: The methodological decision to focus on specific outcomes over a broad definition of self-harm excluded some papers, which have been included in other reviews. It is unlikely to have significantly affected the overall results. The review was not preregistered. CONCLUSIONS: These twin findings highlight the importance overcoming the methodological difficulty of discerning effective interventions during a period of baseline improvement. We suggest that future trials should move away from broadly addressing "suicidality/self-harm" and encourage a greater targeting of at-risk individuals.
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Conducta del Adolescente , Conducta Autodestructiva , Humanos , Adolescente , Ideación Suicida , Intento de SuicidioRESUMEN
Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs.
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Embrión de Mamíferos/metabolismo , Células Endoteliales/metabolismo , Corazón/embriología , Células Madre Pluripotentes/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Embrión de Mamíferos/citología , Células Endoteliales/citología , Humanos , Ratones , Ratones Transgénicos , Células Madre Pluripotentes/citología , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismoRESUMEN
During the initiation of pregnancy, the vasculature of the implantation site expands rapidly, yet little is known about this process or its role in fertility. Here, we report that endothelial-specific deletion of a disintegrin and metalloprotease 10 (ADAM10), an essential regulator of Notch signaling, results in severe subfertility in mice. We found that implantation sites develop until 5.5 days post conception (dpc) but are resorbed by 6.5 dpc in A10ΔEC mice. Analysis of the mutant implantation sites showed impaired decidualization and abnormal vascular patterning compared to controls. Moreover, RNA-seq analysis revealed changes in endothelial cell marker expression consistent with defective ADAM10/Notch signaling in samples from A10ΔEC mice, suggesting that this signaling pathways is essential for the physiological function of endometrial endothelial cells during early pregnancy. Our findings raise the possibility that impaired endothelial cell function could be a cause for repeated pregnancy loss (RPL) and infertility in humans.
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Proteína ADAM10/deficiencia , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Decidua/metabolismo , Fertilidad , Eliminación de Gen , Proteínas de la Membrana/deficiencia , Receptores Notch/metabolismo , Transducción de Señal , Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Femenino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Embarazo , Receptores Notch/genéticaRESUMEN
Marginal integrity is important for the longevity of a restoration. An increase in the marginal discrepancy after cementation contributes adversely to the longevity of a restoration. In the past, the preferred method to overcome this discrepancy was to create internal space for the cement by using a number of coats of a die-spacing material. In the digital age, however, this method is no longer the only option. Currently, an amount of die spacer is engineered into the computer program and forms part of the milling process. The present study attempted to identify the optimal setting of the Spacer parameter that a) is necessary for the complete cementation of a Cerec milled all-ceramic crown, and b) does not compromise the strength of the crown postcementation.
RESUMEN
BACKGROUND: The matrix and associated mesenchyme of the extrahepatic bile ducts are distinct, which could drive diseases with a predilection for these ducts, such as primary sclerosing cholangitis. We aimed to understand the molecular drivers of peribiliary mesenchymal cell (PMC) identity in the extrahepatic bile ducts and dissect how this changed in the context of injury using an entirely in vivo approach with transcriptomic analysis. METHODS AND RESULTS: Single-cell sequencing with a receptor-ligand analysis showed that PMCs had the most interactions with surrounding cells. Wnt4, Wnt5a, and Wnt7b were identified as the major ligands secreted from PMCs and cholangiocytes that interacted in both paracrine and autocrine fashion. Bile duct ligation caused an increase in all 3 Wingless/Integrated ligands and Axin2 with an associated increase in the transcription factors T-box transcription factor (Tbx)2 and Tbx3. Conversely, Indian hedgehog secretion decreased without an associated decrease in hedgehog signaling effectors. Loss of smoothened within PMCs did not impact hedgehog signaling effectors or cellular identity, whereas smoothened gain of function led to myofibroblast transdifferentiation with upregulation of Tbx2 and Tbx3 without injury. Loss of ß-catenin caused a decrease in expression of all 3 Gli transcription factors and associated mesenchymal gene expression, which was phenocopied with compound Gli2 and Gli3 loss in uninjured PMCs. With injury, loss of ß-catenin resulted in decreased myofibroblast transdifferentiation with reduced Tbx2 and Tbx3 expression. CONCLUSIONS: Our results show how modulation of canonical Wingless/Integrated signaling in PMCs is important for regulating basal mesenchymal gene expression and initiating a myogenic gene transcriptional program during injury. They also highlight reciprocating interactions between the hedgehog and Wingless/Integrated signaling pathways within PMCs.
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Proteínas Hedgehog , Vía de Señalización Wnt , Proteínas Hedgehog/genética , Vía de Señalización Wnt/genética , beta Catenina , Homeostasis , Conductos BiliaresRESUMEN
Expanding ß cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain ß cell number. ß cell failure in the IRS2-deletion mouse type 2 diabetes model is, in part, due to loss of CDK4 regulator cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of ß cell mass in IRS2-deficient mice. Surprisingly, not only ß cell mass but also ß cell dedifferentiation was effectively rescued, despite no improvement in whole body insulin sensitivity. Ex vivo studies in primary islet cells revealed a mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical ß cell transcription factor Pdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation and reduced FOXO1 abundance. Taken together, these results demonstrate a differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that ß cell mass can be expanded without compromising function.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Islotes Pancreáticos , Animales , Ratones , Diabetes Mellitus Tipo 2/genética , Diferenciación Celular , Desdiferenciación Celular/genética , Modelos Animales de EnfermedadRESUMEN
Aging associated defects within stem cell-supportive niches contribute towards age-related decline in stem cell activity. However, mechanisms underlying age-related niche defects, and whether restoring niche function can improve stem cell fitness, remain unclear. Here, we sought to determine whether aged blood stem cell function can be restored by rejuvenating their supportive niches within the bone marrow (BM). We identify Netrin-1 as a critical regulator of BM niche cell aging. Niche-specific deletion of Netrin-1 induces premature aging phenotypes within the BM microenvironment, while supplementation of aged mice with Netrin-1 rejuvenates aged niche cells and restores competitive fitness of aged blood stem cells to youthful levels. We show that Netrin-1 plays an essential role in maintaining active DNA damage responses (DDR), and that aging-associated decline in niche-derived Netrin-1 results in DNA damage accumulation within the BM microenvironment. We show that Netrin-1 supplementation is sufficient to resolve DNA damage and restore regenerative potential of the aged BM niche and blood stem cells to endure serial chemotherapy regimens.
Asunto(s)
Médula Ósea , Células Madre Hematopoyéticas , Animales , Ratones , Netrina-1/genética , Células Madre Hematopoyéticas/fisiología , Células de la Médula Ósea , Envejecimiento/genética , Nicho de Células MadreRESUMEN
Glycoengineered bacteria have emerged as a cost-effective platform for rapid and controllable biosynthesis of designer conjugate vaccines. However, little is known about the engagement of such conjugates with naïve B cells to induce the formation of germinal centers (GC), a subanatomical microenvironment that converts naïve B cells into antibody-secreting plasma cells. Using a three-dimensional biomaterials-based B-cell follicular organoid system, we demonstrate that conjugates triggered robust expression of hallmark GC markers, B cell receptor clustering, intracellular signaling, and somatic hypermutation. These responses depended on the relative immunogenicity of the conjugate and correlated with the humoral response in vivo. The occurrence of these mechanisms was exploited for the discovery of high-affinity antibodies against components of the conjugate on a time scale that was significantly shorter than for typical animal immunization-based workflows. Collectively, these findings highlight the potential of synthetic organoids for rapidly predicting conjugate vaccine efficacy as well as expediting antigen-specific antibody discovery.
RESUMEN
BACKGROUND AND OBJECTIVES: COVID-19 progression is characterized by systemic small vessel arterial and venous thrombosis. Microvascular endothelial cell (MVEC) activation and injury, platelet activation, and histopathologic features characteristic of acute COVID-19 also describe certain thrombotic microangiopathies, including atypical hemolytic-uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and hematopoietic stem cell transplant (HSCT)-associated veno-occlusive disease (VOD). We explored the effect of clinically relevant doses of defibrotide, approved for HSCT-associated VOD, on MVEC activation/injury. METHODS: Human dermal MVEC were exposed to plasmas from patients with acute TMAs or acute COVID-19 in the presence and absence of defibrotide (5µg/ml) and caspase 8, a marker of EC activation and apoptosis, was assessed. RNAseq was used to explore potential mechanisms of defibrotide activity. RESULTS: Defibrotide suppressed TMA plasma-induced caspase 8 activation in MVEC (mean 60.2 % inhibition for COVID-19; p = 0.0008). RNAseq identified six major cellular pathways associated with defibrotide's alteration of COVID-19-associated MVEC changes: TNF-α signaling; IL-17 signaling; extracellular matrix (ECM)-EC receptor and platelet receptor interactions; ECM formation; endothelin activity; and fibrosis. Communications across these pathways were revealed by STRING analyses. Forty transcripts showing the greatest changes induced by defibrotide in COVID-19 plasma/MVEC cultures included: claudin 14 and F11R (JAM), important in maintaining EC tight junctions; SOCS3 and TNFRSF18, involved in suppression of inflammation; RAMP3 and transgelin, which promote angiogenesis; and RGS5, which regulates caspase activation and apoptosis. CONCLUSION: Our data, in the context of a recent clinical trial in severe COVID-19, suggest benefits to further exploration of defibrotide and these pathways in COVID-19 and related endotheliopathies.