RESUMEN
Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Hypoxia is a common feature of solid tumors, and cells adapt by upregulating the transcription factor HIF-1α. Here, we defined the transcriptional landscape of mouse tumor-infiltrating natural killer (NK) cells by using single-cell RNA sequencing. Conditional deletion of Hif1a in NK cells resulted in reduced tumor growth, elevated expression of activation markers, effector molecules, and an enriched NF-κB pathway in tumor-infiltrating NK cells. Interleukin-18 (IL-18) from myeloid cells was required for NF-κB activation and the enhanced anti-tumor activity of Hif1a-/- NK cells. Extended culture with an HIF-1α inhibitor increased human NK cell responses. Low HIF1A expression was associated with high expression of IFNG in human tumor-infiltrating NK cells, and an enriched NK-IL18-IFNG signature in solid tumors correlated with increased overall patient survival. Thus, inhibition of HIF-1α unleashes NK cell anti-tumor activity and could be exploited for cancer therapy.
Asunto(s)
Citotoxicidad Inmunológica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Animales , Biomarcadores , Biología Computacional , Citocinas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Activación de Linfocitos/genética , Ratones , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/mortalidad , Pronóstico , Análisis de la Célula Individual , Transcriptoma , Microambiente Tumoral/inmunologíaRESUMEN
BACKGROUND AND AIMS: In hereditary hemorrhagic telangiectasia (HHT), severe liver vascular malformations are associated with mutations in the Activin A Receptor-Like Type 1 ( ACVRL1 ) gene encoding ALK1, the receptor for bone morphogenetic protein (BMP) 9/BMP10, which regulates blood vessel development. Here, we established an HHT mouse model with exclusive liver involvement and adequate life expectancy to investigate ALK1 signaling in liver vessel formation and metabolic function. APPROACH AND RESULTS: Liver sinusoidal endothelial cell (LSEC)-selective Cre deleter line, Stab2-iCreF3 , was crossed with Acvrl1 -floxed mice to generate LSEC-specific Acvrl1 -deficient mice ( Alk1HEC-KO ). Alk1HEC-KO mice revealed hepatic vascular malformations and increased posthepatic flow, causing right ventricular volume overload. Transcriptomic analyses demonstrated induction of proangiogenic/tip cell gene sets and arterialization of hepatic vessels at the expense of LSEC and central venous identities. Loss of LSEC angiokines Wnt2 , Wnt9b , and R-spondin-3 ( Rspo3 ) led to disruption of metabolic liver zonation in Alk1HEC-KO mice and in liver specimens of patients with HHT. Furthermore, prion-like protein doppel ( Prnd ) and placental growth factor ( Pgf ) were upregulated in Alk1HEC-KO hepatic endothelial cells, representing candidates driving the organ-specific pathogenesis of HHT. In LSEC in vitro , stimulation or inhibition of ALK1 signaling counter-regulated Inhibitors of DNA binding (ID)1-3, known Alk1 transcriptional targets. Stimulation of ALK1 signaling and inhibition of ID1-3 function confirmed regulation of Wnt2 and Rspo3 by the BMP9/ALK1/ID axis. CONCLUSIONS: Hepatic endothelial ALK1 signaling protects from development of vascular malformations preserving organ-specific endothelial differentiation and angiocrine signaling. The long-term surviving Alk1HEC-KO HHT model offers opportunities to develop targeted therapies for this severe disease.
Asunto(s)
Telangiectasia Hemorrágica Hereditaria , Ratones , Femenino , Animales , Telangiectasia Hemorrágica Hereditaria/genética , Células Endoteliales/metabolismo , Factor de Crecimiento Placentario/metabolismo , Hígado/patología , Transducción de Señal , Factor 2 de Diferenciación de Crecimiento/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismoRESUMEN
BACKGROUND: Cutaneous melanoma exhibits heterogeneous metastatic patterns and prognosis. In this regard, liver metastasis, which is detected in ~ 10-20% of stage 4 patients, came to the fore of melanoma research, as it recently evolved as decisive indicator of treatment resistance to immune checkpoint inhibition. METHODS: Hepatic metastases were induced by intrasplenic injection of five different murine melanoma cell lines. The efficiencies of hepatic colonization, morphologic patterns, gene expression profiles and degree of vascularization were analyzed and Sorafenib was applied as anti-angiogenic treatment. RESULTS: WT31 melanoma showed the highest efficiency of hepatic colonization, while intermediate efficiencies were observed for B16F10 and RET, and low efficiencies for D4M and HCmel12. RNAseq-based gene expression profiles of high and intermediate metastatic melanomas in comparison to low metastatic melanomas indicated that this efficiency predominantly associates with gene clusters involved in cell migration and angiogenesis. Indeed, heterogeneous vascularization patterns were found in the five models. Although the degree of vascularization of WT31 and B16F10 metastases differed, both showed a strong response to Sorafenib with a successful abrogation of the vascularization. CONCLUSION: Our data indicate that molecular heterogeneity of melanomas can be associated with phenotypic and prognostic features of hepatic metastasis paving the way for organ-specific anti-angiogenic therapeutic approaches.
Asunto(s)
Neoplasias Hepáticas , Melanoma , Neoplasias Cutáneas , Animales , Humanos , Inmunoterapia , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Ratones , Metástasis de la Neoplasia , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND & AIMS: Angiocrine signaling by liver sinusoidal endothelial cells (LSECs) regulates hepatic functions such as growth, metabolic maturation, and regeneration. Recently, we identified GATA4 as the master regulator of LSEC specification during development. Herein, we studied the role of endothelial GATA4 in the adult liver and in hepatic pathogenesis. METHODS: We generated adult Clec4g-icretg/0xGata4fl/fl (Gata4LSEC-KO) mice with LSEC-specific depletion of Gata4. Livers were analyzed by histology, electron microscopy, immunohistochemistry/immunofluorescence, in situ hybridization, and LSECs were isolated for gene expression profiling, ChIP- and ATAC-sequencing. Partial hepatectomy was performed to assess regeneration. We used choline-deficient, l-amino acid-defined (CDAA) diet and chronic carbon tetrachloride exposure to model liver fibrosis. Human single cell RNA-seq data sets were analyzed for endothelial alterations in healthy and cirrhotic livers. RESULTS: Genetic Gata4 deficiency in LSECs of adult mice caused perisinusoidal liver fibrosis, hepatopathy and impaired liver regeneration. Sinusoidal capillarization and LSEC-to-continuous endothelial transdifferentiation were accompanied by a profibrotic angiocrine switch involving de novo endothelial expression of hepatic stellate cell-activating cytokine PDGFB. Increased chromatin accessibility and amplification by activated MYC mediated angiocrine Pdgfb expression. As observed in Gata4LSEC-KO livers, CDAA diet-induced perisinusoidal liver fibrosis was associated with GATA4 repression, MYC activation and a profibrotic angiocrine switch in LSECs. Comparison of CDAA-fed Gata4LSEC-KO and control mice demonstrated that endothelial GATA4 indeed protects against dietary-induced perisinusoidal liver fibrosis. In human cirrhotic livers, GATA4-positive LSECs and endothelial GATA4 target genes were reduced, while non-LSEC endothelial cells and MYC target genes including PDGFB were enriched. CONCLUSIONS: Endothelial GATA4 protects against perisinusoidal liver fibrosis by repressing MYC activation and profibrotic angiocrine signaling at the chromatin level. Therapies targeting the GATA4/MYC/PDGFB/PDGFRß axis offer a promising strategy for prevention and treatment of liver fibrosis. LAY SUMMARY: The liver vasculature is supposed to play a major role in the development of liver fibrosis and cirrhosis, which can lead to liver failure and liver cancer. Herein, we discovered that structural and transcriptional changes induced by genetic deletion of the transcription factor GATA4 in the hepatic endothelium were sufficient to cause liver fibrosis. Activation of the transcription factor MYC and de novo expression of the "angiocrine" growth factor PDGFB were identified as downstream drivers of fibrosis and as potential therapeutic targets for this potentially fatal disease.
Asunto(s)
Células Endoteliales/metabolismo , Factor de Transcripción GATA4/metabolismo , Cirrosis Hepática , Hígado , Linfocinas , Factor de Crecimiento Derivado de Plaquetas , Animales , Cromatina/metabolismo , Descubrimiento de Drogas , Perfilación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/irrigación sanguínea , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/prevención & control , Regeneración Hepática/fisiología , Linfocinas/genética , Linfocinas/metabolismo , Ratones , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Dedos de ZincRESUMEN
Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.
Asunto(s)
Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Cerebelo/patología , Eliminación de Gen , Hipocampo/patología , Proteínas del Tejido Nervioso/deficiencia , Receptores de GABA-B/metabolismo , Animales , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Cerebelo/metabolismo , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Gelatin (GE), amino-functionalized polyphenolic tannin derivative (TN), and graphene oxide (GO) were associated to yield thermo- and pH-responsive hydrogels for the first time. Durable hydrogel assemblies for drug delivery purposes were developed using the photosensitizer methylene blue (MB) as a drug model. The cooling GE/TN blends provide brittle physical assemblies. To overcome this disadvantage, different GO contents (between 0.31% and 1.02% wt/wt) were added to the GE/TN blend at 89.7/10.3 wt/wt. FTIR and RAMAN spectroscopy analyses characterized the materials, indicating GO presence in the hydrogels. Incorporation studies revealed a total MB (0.50 mg/mL) incorporation into the GE/TN-GO hydrogel matrices. Additionally, the proposed systems present a mechanical behavior similar to gel. The GO presence in the hydrogel matrices increased the elastic modulus from 516 to 1650 Pa. SEM revealed that hydrogels containing MB present higher porosity with interconnected pores. Dissolution and swelling degree studies revealed less stability of the GE/TN-GO-MB hydrogels in SGF medium (pH 1.2) than SIF (pH 6.8). The degradation increased in SIF with the GO content, making the polymeric matrices more hydrophilic. MB release studies revealed a process controlled by Fickian diffusion. Our results point out the pH-responsible behavior of mechanically reinforced GE/TN-GO-MB hydrogels for drug delivery systems purposes.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Gelatina/química , Grafito/química , Hidrogeles/química , Azul de Metileno/administración & dosificación , Taninos/química , Temperatura de Transición , Materiales Biocompatibles/química , Difusión , Liberación de Fármacos , Módulo de Elasticidad , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Transición de Fase , PorosidadRESUMEN
Leda-1/Pianp is a type I transmembrane protein expressed by CNS cells, murine melanoma cell line B16F10 and rat liver sinusoidal endothelial cells. The early steps of posttranslational modifications of Leda-1/Pianp have been described to include glycosylation and processing by proprotein convertases. Here, we comprehensively characterized the subsequent steps of proteolytic processing of Leda-1/Pianp. For this purpose specific protease inhibitors and cell lines deficient in PS1, PS2, PS1/PS2 and ADAM10/17 were deployed. Leda-1/Pianp was cleaved at numerous cleavage sites within the N-terminal extracellular domain. The sheddases involved included MMPs and ADAM10/17. Ectodomain shedding yielded C-terminal fragments (CTF) of â¼15 kDa. The CTF was further processed by the γ (gamma)-secretase complex to generate the intracellular domain (ICD) of â¼10 kDa. Although PS1 was the dominant intramembrane protease, PS2 was also able to cleave Leda-1/Pianp in the absence of PS1. Thus, Leda-1/Pianp is constitutively processed by proprotein convertases, sheddases including MMPs and ADAM10/17 and intramembrane protease γ-secretase.
Asunto(s)
Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetulus , Activación Enzimática , Células HEK293 , Humanos , Ratones , Unión Proteica , Procesamiento Proteico-Postraduccional/fisiología , Proteolisis , Especificidad por SustratoRESUMEN
Leda-1/Pianp is a type-I transmembrane protein which is sorted to the basolateral membrane domain of polarized epithelial cells. Here, we investigated trafficking mechanisms and functions of Leda-1/Pianp in MDCK and MCF-7 cells. Basolateral sorting and posttranslational modifications depended on the intracellular juxtamembrane region. Functionally, Leda-1/Pianp increased the transepithelial electrical resistance generated by a polarized cell sheet. Furthermore, resistance to junctional destabilization by tumor cells was enhanced by Leda-1/Pianp indicating increased stability and tightness of intercellular junctions. While Claudin 1 and 4 expression and activities of small GTPases were not affected, γ-Secretase-mediated cleavage of E-Cadherin was attenuated by Leda-1/Pianp. Regulation of proteolytic processing is thus a molecular mechanism by which Leda-1/Pianp can affect junctional integrity and function.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cadherinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Antígenos CD , Perros , Impedancia Eléctrica , Células Epiteliales/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Células de Riñón Canino Madin DarbyRESUMEN
Liver endothelial differentiation-associated protein-1 (Leda-1/Pianp) is a type-I-transmembrane protein that is able to bind and activate immune inhibitory receptor Pilrα. Here we show that Leda-1/Pianp is strain-specifically expressed in lymphoid organs and macrophages of Th2-prone BALB/c mice but not of Th1-prone C57BL/6J mice. LPS stimulation of BALB/c bone marrow-derived macrophages (BMM) and macrophage-like Raw 264.7 cells conversely regulated Leda-1/Pianp and Pilrα expression. Pilrα induction was caused by LPS-mediated transcriptional modulation and increased mRNA expression. On the other hand, the LPS-mediated decline of Leda-1/Pianp expression was the result of proteolytic degradation by matrix metalloproteinases. In summary, these findings demonstrate that counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα is part of the complex innate immune response of macrophages and its genetically determined strain-specific modulation.
Asunto(s)
Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Inmunidad Innata/genética , Ligandos , Lipopolisacáridos/farmacología , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Activación de Macrófagos , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Especificidad de la EspecieRESUMEN
Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL, LT) is a subtype of cutaneous B-cell lymphoma with unfavorable prognosis usually requiring aggressive polychemotherapy for disease control. Only single cases of spontaneous regression of PCDLBCL, LT are reported in the literature, peaking 3 months post-biopsy following a clinical history of no longer than 1 year. Here, we report the first case of a spontaneously relapsing and remitting PCDLBCL, LT with complete regression after a clinical history of more than 9 years and thus an atypically indolent clinical course. The female patient presented with recurrent erythematous, non-ulcerated, non-raised plaques of the right lower leg for 6 years. Pathological workup and exclusion of a systemic disease confirmed the diagnosis of PCDLBCL, LT. Due to the history of repeated spontaneous remission, no therapy was initiated. Nine years after first occurrence the patient presented with complete clinical remission lasting for 64 months. We retrospectively identified four additional PCDLBCL, LT patients with spontaneous remission lasting up to 53 months. Our data provide evidence for a distinct PCDLBCL, LT patient subgroup that clinicians should be aware of and warrants a watch-and-wait treatment regime.
RESUMEN
Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.
Asunto(s)
Insuficiencia Cardíaca , Factores de Transcripción , Animales , Humanos , Ratones , Modelos Animales de Enfermedad , Células Endoteliales , Fibrosis , Péptidos y Proteínas de Señalización Intercelular , Cirrosis Hepática/complicaciones , Factor de Transcripción SOX9/genéticaRESUMEN
Liver endothelial differentiation-associated protein-1 (LEDA-1/PIANP) is a type-I-transmembrane protein first identified by us as a putative junctional protein in liver sinusoidal endothelial cells. Others have shown that LEDA-1/PIANP binds and activates immune inhibitory receptor PILRα in trans, a process that requires sialidation of LEDA-1/PIANP. Here we show that LEDA-1/PIANP is subject to O-glycosylation and sialidation as demonstrated in brain tissue as well as in LEDA-1 expressing cell lines by using anti-LEDA-1/PIANP C-terminal antibodies. In addition, analysis of LEDA-1/PIANP processing with His-tags inserted at different positions in the extracellular domain revealed that multiple steps of proteolytic cleavage occur during maturation of the protein. Proteolytic cleavage between aa59 and aa83 preceded sorting of the protein to the plasma membrane. Deletion of aa75-79 and inhibition with Furin inhibitor I confirmed that LEDA-1/PIANP is processed by a Furin-like proprotein convertase. In summary, these findings show that Furin-like proprotein convertase-dependent processing precedes plasma membrane localization of LEDA-1/PIANP that is a pre-requisite of functional receptor-ligand interactions in vitro and in vivo.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proproteína Convertasas/metabolismo , Proteolisis , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Perros , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Furina/metabolismo , Células HEK293 , Humanos , Ligandos , Ratones , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Mapeo de Interacción de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
Machine learning algorithms provide detailed description of the anaerobic digestion process, but the impact of data preparation procedures and hyperparameter optimization has rarely been investigated. A genetic algorithm was developed for optimizing data preparation and model hyperparameters to simulate dynamic methane production from steady-state anaerobic digestion of agricultural residues at full-scale. A long short-term memory neural network was used as prediction model. Results indicate that batch size, learning rate and number of neurons are the most important model parameters for accurate description of methane production rates, whereas combination of hyperparameter and data preparation optimization shows best model efficiencies, with a root mean square scaled error of 76.5 %. Mass of solid feed, time and mass of volatile solids are the most relevant input features. This study provides fundamental steps for optimal prediction of dynamic biomethane production, as a reliable basis for improving bioconversion efficiency during anaerobic digestion of agricultural residues.
Asunto(s)
Reactores Biológicos , Metano , Anaerobiosis , Biocombustibles , Aprendizaje AutomáticoRESUMEN
Obtaining CO and H2 from electrochemical CO2 reduction (CO2RR) offers a viable alternative to reduce CO2 emissions and produce chemicals and fuels. Herein, we report a simple strategy for obtaining polycrystalline copper deposited on oxidized graphite felt (Cu-OGF) and its performance on the selective conversion of CO2 and H2O to CO and H2. For the electrode obtaining, graphite felt (GF) was first oxidized (OGF) in order to make the substrate hydrophilic and then copper particles were electrochemically deposited onto OGF. The pH of deposition was investigated, and the CO2RR activity was assessed for the prepared electrodes at each pH (2.0, 4.0, 6.0, 8.0, and 10.0). It was found that pH 2.0 was the most promising for CO2RR due to the presence of hexagonal copper microparticles. Fourier transform infrared analysis of the produced gases showed that this is a low-cost catalyst capable of reducing CO2 and H2O to CO and H2, with Faradaic efficiencies between 0.50 and 5.21% for CO and 50.87 to 98.30% for H2, depending on the experimental conditions. Hence, it is possible for this gas mixture to be used as a fuel gas or to be enriched with CO for use in Fischer-Tropsch processes.
RESUMEN
BACKGROUND: Open pneumothorax after a penetrating thorax trauma is a life-threatening disease with high mortality. An emergency application of a chest seal (CS) allowing the release of trapped air is the optimum initial therapy until surgical chest drainage is available. METHODS: In a newly developed experimental porcine model of open pneumothorax, we tested 3 different materials regarding their applicability for acute treatment of sucking chest wounds in prehospital emergency care, namely a commonly used rescue blanket (RB), plastic foil from a gauze package (packing material), and a commercial CS. RESULTS: An ex vivo open pneumothorax model using a porcine chest wall and a vacuum-assisted drainage system was successfully established. RB segments sized 70 × 100 mm achieved significantly higher rates of successful sealing than plastic foils from a gauze package sized 100 × 100 mm when the devices were applied to the moistened chest wall and fixed on 3 sides (5/5 [100%] vs 0/5 [0%], respectively; P = .002). Loosely fixed RBs efficiently released injected air (10/10 [100%]) and consequently sealed the wound in all cases (10/10). CONCLUSIONS: RBs, applied wet, are appropriate CSs with good occlusive and adherence properties. Fixation on 2 sides of the dressing is sufficient to allow trapped air to exit while providing appropriate sealing of the chest wound. RBs were superior to plastic foils from a gauze package and were seen to function as a potent makeshift CS when no commercial CS is available.
Asunto(s)
Neumotórax , Traumatismos Torácicos , Heridas Penetrantes , Animales , Humanos , Plásticos , Neumotórax/terapia , Porcinos , Traumatismos Torácicos/cirugía , TóraxRESUMEN
The Class H scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two of the most highly expressed genes in liver sinusoidal endothelial cells (LSECs). While Stab1-deficient (Stab1KO) and Stab2-deficient (Stab2KO) mice are phenotypically unremarkable, Stab1/2-double-deficient (StabDKO) mice exhibit perisinusoidal liver fibrosis, glomerulofibrotic nephropathy and a reduced life expectancy. These conditions are caused by insufficiently scavenged circulating noxious blood factors. The effects of either Stab-single- or double-deficiency on LSEC differentiation and function, however, have not yet been thoroughly investigated. Therefore, we performed comprehensive transcriptomic analyses of primary LSECs from Stab1KO, Stab2KO and StabDKO mice. Microarray analysis revealed dysregulation of pathways and genes involved in established LSEC functions while sinusoidal endothelial marker gene expression was grossly unchanged. 82 genes were significantly altered in Stab1KO, 96 genes in Stab2KO and 238 genes in StabDKO compared with controls; 42 genes were found to be commonly dysregulated in all three groups and all of these genes were downregulated. These commonly downregulated genes (CDGs) were categorized as "potential scavengers," "cell adhesion molecules," "TGF-ß/BMP-signaling" or "collagen and extracellular matrix (ECM) components". Among CDGs, Colec10, Lumican and Decorin, were the most strongly down-regulated genes and the corresponding proteins impact on the interaction of LSECs with chemokines, ECM components and carbohydrate structures. Similarly, "chemokine signaling," "cytokine-cytokine receptor interaction" and "ECM-receptor interaction," were the GSEA categories which represented most of the downregulated genes in Stab1KO and Stab2KO LSECs. In summary, our data show that loss of a single Stabilin scavenger receptor - and to a greater extent of both receptors - profoundly alters the transcriptomic repertoire of LSECs. These alterations may affect LSEC-specific functions, especially interactions of LSECs with the ECM and during inflammation as well as clearance of the peripheral blood.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/genética , Cirrosis Hepática/genética , Animales , Moléculas de Adhesión Celular Neuronal/deficiencia , Quimiocinas/metabolismo , Perfilación de la Expresión Génica , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transcriptoma/genéticaRESUMEN
Endothelial wingless-related integration site (Wnt)-/ß-catenin signaling is a key regulator of the tightly sealed blood-brain barrier. In the hepatic vascular niche angiokine-mediated Wnt signaling was recently identified as an important regulator of hepatocyte function, including the determination of final adult liver size, liver regeneration, and metabolic liver zonation. Within the hepatic vasculature, the liver sinusoidal endothelial cells (LSECs) are morphologically unique and functionally specialized microvascular endothelial cells (ECs). Pathological changes of LSECs are involved in chronic liver diseases, hepatocarcinogenesis, and liver metastasis. To comprehensively analyze the effects of endothelial Wnt-/ß-catenin signaling in the liver, we used endothelial subtype-specific Clec4g-iCre mice to generate hepatic ECs with overexpression of Ctnnb1. In the resultant Clec4g-iCre tg/wt ;Ctnnb1(Ex3) fl/wt (Ctnnb1 OE-EC ) mice, activation of endothelial Wnt-/ß-catenin signaling resulted in sinusoidal transdifferentiation with disturbed endothelial zonation, that is, loss of midzonal LSEC marker lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve1) and enrichment of continuous EC genes, such as cluster of differentiation (CD)34 and Apln. Notably, gene set enrichment analysis revealed overrepresentation of brain endothelial transcripts. Activation of endothelial Wnt-/ß-catenin signaling did not induce liver fibrosis or alter metabolic liver zonation, but Ctnnb1 OE-EC mice exhibited significantly increased plasma triglyceride concentrations, while liver lipid content was slightly reduced. Ctnnb1 overexpression in arterial ECs of the heart has been reported previously to cause cardiomyopathy. As Clec4g-iCre is active in a subset of cardiac ECs, it was not unexpected that Ctnnb1 OE-EC mice showed reduced overall survival and cardiac dysfunction. Altogether, balanced endothelial Wnt-/ß-catenin signaling in the liver is required for normal LSEC differentiation and for maintenance of normal plasma triglyceride levels.
RESUMEN
Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized ß-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII-PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.
Asunto(s)
Anemia/genética , Médula Ósea/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Endotelio Vascular/metabolismo , Eritroblastos/metabolismo , Eritropoyesis/genética , beta Catenina/genética , Anemia/metabolismo , Anemia/mortalidad , Anemia/patología , Animales , Médula Ósea/irrigación sanguínea , Capilares/citología , Capilares/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Diferenciación Celular , Células Endoteliales/clasificación , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Eritroblastos/clasificación , Eritroblastos/citología , Femenino , Factor-23 de Crecimiento de Fibroblastos/genética , Factor-23 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Integrasas/genética , Integrasas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Ratones Transgénicos , Osteogénesis , Reticulocitos/citología , Reticulocitos/metabolismo , Análisis de Supervivencia , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Pseudomonas fluorescens strains are used in agriculture as plant growth-promoting rhizobacteria (PGPR). Nontarget effects of released organisms should be analyzed prior to their large-scale use, and methods should be available to sensitively detect possible changes in the environments the organism is released to. According to ecological theory, microbial communities with a greater diversity should be less susceptible to disturbance by invading organisms. Based on this principle, we laid out a pot experiment with field-derived soils different in their microbial biomass and activity due to long-term management on similar parent geological material (loess). We investigated the survival of P. fluorescens CHA0 that carried a resistance toward rifampicin and the duration of potential changes of the soil microflora caused by the inoculation with the bacterium at the sowing date of spring wheat. Soil microbial biomass (C(mic), N(mic)) basal soil respiration (BR), qCO(2), dehydrogenase activity (DHA), bacterial plate counts, mycorrhiza root colonization, and community level substrate utilization were analyzed after 18 and 60 days. At the initial stage, soils were clearly different with respect to most of the parameters measured, and a time-dependent effect between the first and the second set point were attributable to wheat growth and the influence of roots. The effect of the inoculum was small and merely transient, though significant long-term changes were found in soils with a relatively low level of microbial biomass. Community level substrate utilization as an indicator of changes in microbial community structure was mainly changed by the growth of wheat, while other experimental factors were negligible. The sensitivity of the applied methods to distinguish the experimental soils was in decreasing order N(mic), DHA, C(mic), and qCO(2). Besides the selective enumeration of P. fluorescens CHA0 rif(+), which was only found in amended soils, methods to distinguish the inoculum effect were DHA, C(mic), and the ratio of C(mic) to N(mic). The sampling time was most sensitively indicated by N(mic), DHA, C(mic), and qCO(2). Our data support the hypothesis-based on ecosystem theory-that a rich microflora is buffering changes due to invading species. In other words, a soil-derived bacterium was more effective in a relatively poor soil than in soils that are rich in microorganisms.
Asunto(s)
Agricultura/métodos , Biomasa , Pseudomonas fluorescens/crecimiento & desarrollo , Microbiología del Suelo , Suelo/análisis , Recuento de Colonia Microbiana , Productos Agrícolas/microbiología , Ecosistema , Raíces de Plantas/microbiología , Pseudomonas fluorescens/metabolismo , Triticum/microbiologíaRESUMEN
The interaction of tumor cells with organ-specific endothelial cells (EC) is an important step during metastatic progression. Notch signaling in organ-specific niches has been implicated in mediating opposing effects on organotropic metastasis to the lungs and the liver, respectively. In this study, we scrutinized the role of endothelial Notch activation during liver metastasis. To target hepatic EC (HEC), a novel EC subtype-specific Cre driver mouse was generated. Clec4g-Cretg/wt mice were crossed to Rosa26N1ICD-IRES-GFP to enhance Notch signaling in HEC (NICDOE-HEC). In NICDOE-HEC mice, hepatic metastasis of malignant melanoma and colorectal carcinoma was significantly reduced. These mice revealed reduced liver growth and impaired metabolic zonation due to suppression of hepatic angiocrine Wnt signaling. Hepatic metastasis, however, was not controlled by angiocrine Wnt signaling, as deficiency of the Wnt cargo receptor Wls in HEC of WlsHEC-KO mice did not affect hepatic metastasis. In contrast, the hepatic microvasculature in NICDOE-HEC mice revealed a special form of sinusoidal capillarization, with effacement of endothelial zonation functionally paralleled by reduced tumor cell adhesion in vivo. Notably, expression of endothelial adhesion molecule ICAM1 by HEC was significantly reduced. Treatment with an anti-ICAM1 antibody significantly inhibited tumor cell adhesion to HEC in wild-type mice confirming that Notch controls hepatic metastasis via modulation of HEC adhesion molecules. As endothelial Notch activation in the lung has been shown to promote lung metastasis, tumor therapy will require approaches that target Notch in an organ-, cell type-, and context-specific manner. SIGNIFICANCE: Manipulation of Notch signaling in the endothelium has opposing, organ-specific effects on metastasis to the lung and the liver, demonstrating that this pathway should be targeted in a cell- and context-specific fashion.