RESUMO
ETS transcription factors ETV2, FLI1, and ERG1 specify pluripotent stem cells into induced vascular endothelial cells (iVECs). However, iVECs are unstable and drift toward nonvascular cells. We show that human midgestation c-Kit(-) lineage-committed amniotic cells (ACs) can be reprogrammed into vascular endothelial cells (rAC-VECs) without transitioning through a pluripotent state. Transient ETV2 expression in ACs generates immature rAC-VECs, whereas coexpression with FLI1/ERG1 endows rAC-VECs with a vascular repertoire and morphology matching mature endothelial cells (ECs). Brief TGFß-inhibition functionalizes VEGFR2 signaling, augmenting specification of ACs into rAC-VECs. Genome-wide transcriptional analyses showed that rAC-VECs are similar to adult ECs in which vascular-specific genes are expressed and nonvascular genes are silenced. Functionally, rAC-VECs form stable vasculature in Matrigel plugs and regenerating livers. Therefore, short-term ETV2 expression and TGFß inhibition with constitutive ERG1/FLI1 coexpression reprogram mature ACs into durable rAC-VECs with clinical-scale expansion potential. Banking of HLA-typed rAC-VECs establishes a vascular inventory for treatment of diverse disorders.
Assuntos
Líquido Amniótico/citologia , Diferenciação Celular , Células Endoteliais/citologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , HumanosRESUMO
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.
Assuntos
Antivirais/farmacologia , COVID-19/virologia , Colo/citologia , Avaliação Pré-Clínica de Medicamentos/métodos , Pulmão/citologia , Organoides/efeitos dos fármacos , Organoides/virologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/prevenção & controle , Colo/efeitos dos fármacos , Colo/virologia , Aprovação de Drogas , Feminino , Xenoenxertos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/virologia , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , SARS-CoV-2/genética , Estados Unidos , United States Food and Drug Administration , Tropismo Viral , Internalização do Vírus/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
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.
Assuntos
Vasos Sanguíneos/citologia , Carcinogênese , Células Endoteliais/citologia , Hemodinâmica , Neoplasias/irrigação sanguínea , Organogênese , Organoides/irrigação sanguínea , Vasos Sanguíneos/crescimento & desenvolvimento , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Cromatina/metabolismo , Epigênese Genética , Epigenômica , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Ilhotas Pancreáticas/irrigação sanguínea , Modelos Biológicos , Especificidade de Órgãos , RNA-Seq , Análise de Célula Única , Fatores de Transcrição , TranscriptomaRESUMO
Stroke-induced cerebral microvascular dysfunction contributes to aggravation of neuronal injury and compromises the efficacy of current reperfusion therapies. Understanding the molecular alterations in cerebral microvessels in stroke will provide original opportunities for scientific investigation of novel therapeutic strategies. Toward this goal, using a recently optimized method which minimizes cell activation and preserves endothelial cell interactions and RNA integrity, we conducted a genome-wide transcriptomic analysis of cerebral microvessels in a mouse model of stroke and compared these transcriptomic alterations with the ones observed in human, nonfatal, brain stroke lesions. Results from these unbiased comparative analyses have revealed the common alterations in mouse stroke microvessels and human stroke lesions and identified shared molecular features associated with vascular disease (e.g., Serpine1/Plasminogen Activator Inhibitor-1, Hemoxygenase-1), endothelial activation (e.g., Angiopoietin-2), and alterations in sphingolipid metabolism and signaling (e.g., Sphigosine-1-Phosphate Receptor 2). Sphingolipid profiling of mouse cerebral microvessels validated the transcript data and revealed the enrichment of sphingomyelin and sphingoid species in the cerebral microvasculature compared to brain and the stroke-induced increase in ceramide species. In summary, our study has identified novel molecular alterations in several microvessel-enriched, translationally relevant, and druggable targets, which are potent modulators of endothelial function. Our comparative analyses have revealed the presence of molecular features associated with cerebral microvascular dysfunction in human chronic stroke lesions. The results shared here provide a detailed resource for therapeutic discovery of candidates for neurovascular protection in stroke and potentially, other pathologies exhibiting cerebral microvascular dysfunction.
Assuntos
Acidente Vascular Cerebral , Camundongos , Humanos , Animais , Acidente Vascular Cerebral/metabolismo , Encéfalo/metabolismo , Endotélio/metabolismo , Microvasos/patologia , Esfingolipídeos/metabolismo , Barreira Hematoencefálica/metabolismoRESUMO
Peripheral T-cell lymphomas (PTCL) with T-follicular helper phenotype (PTCL-TFH) has recurrent mutations affecting epigenetic regulators, which may contribute to aberrant DNA methylation and chemoresistance. This phase 2 study evaluated oral azacitidine (CC-486) plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) as initial treatment for PTCL. CC-486 at 300 mg daily was administered for 7 days before C1 of CHOP, and for 14 days before CHOP C2-6. The primary end point was end-of-treatment complete response (CR). Secondary end points included safety and survival. Correlative studies assessed mutations, gene expression, and methylation in tumor samples. Grade 3 to 4 hematologic toxicities were mostly neutropenia (71%), with febrile neutropenia uncommon (14%). Nonhematologic toxicities included fatigue (14%) and gastrointestinal symptoms (5%). In 20 evaluable patients, CR was 75%, including 88.2% for PTCL-TFH (n = 17). The 2-year progression-free survival (PFS) was 65.8% for all and 69.2% for PTCL-TFH, whereas 2-year overall survival (OS) was 68.4% for all and 76.1% for PTCL-TFH. The frequencies of the TET2, RHOA, DNMT3A, and IDH2 mutations were 76.5%, 41.1%, 23.5%, and 23.5%, respectively, with TET2 mutations significantly associated with CR (P = .007), favorable PFS (P = .004) and OS (P = .015), and DNMT3A mutations associated with adverse PFS (P = .016). CC-486 priming contributed to the reprograming of the tumor microenvironment by upregulation of genes related to apoptosis (P < .01) and inflammation (P < .01). DNA methylation did not show significant shift. This safe and active regimen is being further evaluated in the ALLIANCE randomized study A051902 in CD30-negative PTCL. This trial was registered at www.clinicaltrials.gov as #NCT03542266.
Assuntos
Linfoma de Células T Periférico , Humanos , Linfoma de Células T Periférico/patologia , Azacitidina/efeitos adversos , Doxorrubicina , Prednisona/efeitos adversos , Vincristina , Ciclofosfamida/efeitos adversos , Fatores Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Microambiente TumoralRESUMO
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.
Assuntos
Endotélio Vascular/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/genética , Animais , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/fisiologia , Endotélio Vascular/fisiologia , Expressão Gênica , Humanos , Camundongos Endogâmicos , Células-Tronco Pluripotentes/fisiologia , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Análise de Célula Única , Fatores de Transcrição/metabolismo , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismoRESUMO
[Figure: see text].
Assuntos
COVID-19/imunologia , Imunidade Celular/imunologia , Mediadores da Inflamação/imunologia , Macrófagos/imunologia , Miócitos Cardíacos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/patologia , Técnicas de Cocultura/métodos , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Miocárdio/imunologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Células-Tronco Pluripotentes/imunologia , Análise de Sequência de RNA/métodosRESUMO
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.
Assuntos
Diferenciação Celular , Reprogramação Celular , Endotélio/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Imunidade Adaptativa , Envelhecimento/genética , Animais , Linhagem Celular , Linhagem da Célula , Autorrenovação Celular , Células Clonais/citologia , Células Clonais/transplante , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
The preservation of labile biomolecules presents a major challenge in chemistry, and deep eutectic solvents (DESs) have emerged as suitable environments for this purpose. However, how the hydration of DESs impacts the behavior of proteins is often neglected. Here, we demonstrate that the amino acid environment and secondary structure of two proteins (bovine serum albumin and lysozyme) and an antibody (immunoglobulin G) in 1:2 choline chloride:glycerol and 1:2 choline chloride:urea follow a re-entrant behavior with solvent hydration. A dome-shaped transition is observed with a folded or partially folded structure at very low (<10 wt % H2O) and high (>40 wt % H2O) DES hydration, while protein unfolding increases between those regimes. Hydration also affects protein conformation and stability, as demonstrated for bovine serum albumin in hydrated 1:2 choline chloride:glycerol. In the neat DES, bovine serum albumin remains partially folded and unexpectedly undergoes unfolding and oligomerization at low water content. At intermediate hydration, the protein begins to refold and gradually retrieves the native monomer-dimer equilibrium. However, ca. 36 wt % H2O is required to recover the native folding fully. The half-denaturation temperature of the protein increases with decreasing hydration, but even the dilute DESs significantly enhance the thermal stability of bovine serum albumin. Also, protein unfolding can be reversed by rehydrating the sample to the high hydration regime, also recovering protein function. This correlation provides a new perspective to understanding protein behavior in hydrated DESs, where quantifying the DES hydration becomes imperative to identifying the folding and stability of proteins.
Assuntos
Solventes Eutéticos Profundos , Glicerol , Soroalbumina Bovina/química , Solventes/química , ColinaRESUMO
MOTIVATION: The use of liquid biopsies for cancer patients enables the non-invasive tracking of treatment response and tumor dynamics through single or serial blood drawn tests. Next-generation sequencing assays allow for the simultaneous interrogation of extended sets of somatic single-nucleotide variants (SNVs) in circulating cell-free DNA (cfDNA), a mixture of DNA molecules originating both from normal and tumor tissue cells. However, low circulating tumor DNA (ctDNA) fractions together with sequencing background noise and potential tumor heterogeneity challenge the ability to confidently call SNVs. RESULTS: We present a computational methodology, called Adaptive Base Error Model in Ultra-deep Sequencing data (ABEMUS), which combines platform-specific genetic knowledge and empirical signal to readily detect and quantify somatic SNVs in cfDNA. We tested the capability of our method to analyze data generated using different platforms with distinct sequencing error properties and we compared ABEMUS performances with other popular SNV callers on both synthetic and real cancer patients sequencing data. Results show that ABEMUS performs better in most of the tested conditions proving its reliability in calling low variant allele frequencies somatic SNVs in low ctDNA levels plasma samples. AVAILABILITY AND IMPLEMENTATION: ABEMUS is cross-platform and can be installed as R package. The source code is maintained on Github at http://github.com/cibiobcg/abemus, and it is also available at CRAN official R repository. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Nucleotídeos , Reprodutibilidade dos TestesRESUMO
The incidence of pancreatic neuroendocrine tumor (PNET) is increasing, and it presents with various clinical manifestations and an unfavorable survival rate. A better understanding of the drivers of PNET tumorigenesis is urgently needed. Distinct miRNA signatures have been identified for different stages of tumorigenesis in both human and mouse PNETs. The functions of these miRNAs are poorly understood. miR-431 is the most up-regulated miRNA in the metastatic signature. However, it is unknown whether miR-431 contributes to metastasis of PNETs. Herein, we show that miR-431 overexpression activates Ras/extracellular signal-regulated kinase (Erk) signaling and promotes epithelial-mesenchymal transition, migration/invasion in vitro, and metastasis in both xenograft and spontaneous mouse models of PNET. Treatment of PNET cells with Erk inhibitor or locked nucleic acids sequestering miR-431 inhibits invasion. Four target prediction modules and dual-luciferase reporter assays were used to identify potential mRNA targets of miR-431. A Ras GTPase activating protein tumor suppressor (RasGAP), DAB2 interacting protein (DAB2IP), was discovered as an miR-431 target. Overexpression of DAB2IP's rat homolog, but not its mutant defective in Ras GTPase activating protein activity, reverses miR-431's effect on promoting invasion, Erk phosphorylation, and epithelial-mesenchymal transition of PNETs. Taken together, miR-431 silences DAB2IP to active Ras/Erk and promote metastasis of PNETs. miR-431 may be targeted to manage metastatic PNETs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/genética , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Carcinogênese , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Metástase Neoplásica , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Ratos , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
BACKGROUND: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. METHODS: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. RESULTS: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. CONCLUSIONS: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.
Assuntos
Perfilação da Expressão Gênica , Rejeição de Enxerto/sangue , Rejeição de Enxerto/diagnóstico , Falência Renal Crônica/cirurgia , Transplante de Rim/efeitos adversos , Adulto , Idoso , Biomarcadores/metabolismo , Biópsia , Feminino , Genômica , Sobrevivência de Enxerto , Humanos , Imunossupressores/uso terapêutico , Inflamação , Estimativa de Kaplan-Meier , Falência Renal Crônica/sangue , Falência Renal Crônica/mortalidade , Transplante de Rim/mortalidade , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Estudos Prospectivos , Fatores de Risco , Análise de Sequência de RNARESUMO
BACKGROUND: Schistosomiasis increases the risk of human immunodeficiency virus (HIV) acquisition in women by mechanisms that are incompletely defined. Our objective was to determine how the cervical environment is impacted by Schistosoma haematobium or Schistosoma mansoni infection by quantifying gene expression in the cervical mucosa and cytokine levels in cervicovaginal lavage fluid. METHODS: We recruited women with and those without S. haematobium infection and women with and those without S. mansoni infection from separate villages in rural Tanzania with high prevalences of S. haematobium and S. mansoni, respectively. Infection status was determined by urine and stool microscopy and testing for serum circulating anodic antigen. RNA was extracted from cervical cytobrush samples for transcriptome analysis. Cytokine levels were measured by magnetic bead immunoassay. RESULTS: In the village where S. haematobium was prevalent, 110 genes were differentially expressed in the cervical mucosa of 18 women with versus 39 without S. haematobium infection. Among the 27 cytokines analyzed in cervicovaginal lavage fluid from women in this village, the level of interleukin 15 was lower in the S. haematobium-infected group (62.8 vs 102.9 pg/mL; adjusted P = .0013). Differences were not observed in the S. mansoni-prevalent villages between 11 women with and 29 without S. mansoni infection. CONCLUSIONS: We demonstrate altered cervical mucosal gene expression and lower interleukin 15 levels in women with S. haematobium infection as compared to those with S. mansoni infection, which may influence HIV acquisition and cancer risks. Studies to determine the effects of antischistosome treatment on these mucosal alterations are needed.
Assuntos
Interleucina-15/genética , Schistosoma haematobium/imunologia , Schistosoma mansoni/imunologia , Esquistossomose Urinária/imunologia , Esquistossomose mansoni/imunologia , Adulto , Animais , Feminino , Humanos , Mucosa/imunologia , Mucosa/parasitologia , Prevalência , População Rural , Esquistossomose Urinária/epidemiologia , Esquistossomose Urinária/parasitologia , Esquistossomose mansoni/epidemiologia , Esquistossomose mansoni/parasitologia , Tanzânia/epidemiologia , Adulto JovemRESUMO
Schistosome worms infect over 200 million people worldwide. They live in the host's bloodstream and alter host immunity. Epidemiological data suggest that males and females have different responses to schistosome infection, but the effect of sex on systemic response is undetermined. Our objective was to characterize differences in peripheral blood transcriptional profiles in people with or without active Schistosoma haematobium infection and to determine whether this signature differs between males and females. mRNA was isolated using poly(A) selection and sequenced on an Illumina Hi-Seq4000 platform. Transcripts were aligned to the human hg19 reference genome and counted with the HTSeq package. Genes were compared for differential expression using DESeq2. Ingenuity Pathway Analysis (IPA) was used to identify gene networks altered in the presence of S. haematobium We enrolled 33 participants from villages in rural Tanzania where S. haematobium is endemic. After correction for multiple comparisons, we observed 383 differentially expressed genes between those with or without S. haematobium infection when sex was included as a covariate. Heat-mapping of the genes with >1.5-fold differences in gene expression revealed clustering by S. haematobium infection status. The top networks included development, cell death and survival, cell signaling, and immunologic disease pathways. We observed a distinct whole blood transcriptional profile, as well as differences in men and women, with S. haematobium infection. Additional studies are needed to determine the clinical effects of these divergent responses. Attention to sex-based differences should be included in studies of human schistosome infection.
Assuntos
Células Sanguíneas/imunologia , Células Sanguíneas/parasitologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Schistosoma haematobium/imunologia , Esquistossomose Urinária/imunologia , Esquistossomose Urinária/patologia , Adolescente , Adulto , Animais , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Schistosoma haematobium/crescimento & desenvolvimento , Análise de Sequência de RNA , Fatores Sexuais , Tanzânia , Adulto JovemRESUMO
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy caused by mutations in PKD1 and PKD2 that is characterized by renal tubular epithelial cell proliferation and progressive CKD. Although the molecular mechanisms involved in cystogenesis are not established, concurrent inactivating constitutional and somatic mutations in ADPKD genes in cyst epithelium have been proposed as a cellular recessive mechanism. METHODS: We characterized, by whole-exome sequencing (WES) and long-range PCR techniques, the somatic mutations in PKD1 and PKD2 genes in renal epithelial cells from 83 kidney cysts obtained from nine patients with ADPKD, for whom a constitutional mutation in PKD1 or PKD2 was identified. RESULTS: Complete sequencing data by long-range PCR and WES was available for 63 and 65 cysts, respectively. Private somatic mutations of PKD1 or PKD2 were identified in all patients and in 90% of the cysts analyzed; 90% of these mutations were truncating, splice site, or in-frame variations predicted to be pathogenic mutations. No trans-heterozygous mutations of PKD1 or PKD2 genes were identified. Copy number changes of PKD1 ranging from 151 bp to 28 kb were observed in 12% of the cysts. WES also identified significant mutations in 53 non-PKD1/2 genes, including other ciliopathy genes and cancer-related genes. CONCLUSIONS: These findings support a cellular recessive mechanism for cyst formation in ADPKD caused primarily by inactivating constitutional and somatic mutations of PKD1 or PKD2 in kidney cyst epithelium. The potential interactions of these genes with other ciliopathy- and cancer-related genes to influence ADPKD severity merits further evaluation.
Assuntos
Células Epiteliais/metabolismo , Transplante de Rim/métodos , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/cirurgia , Canais de Cátion TRPP/genética , Adulto , Proliferação de Células/genética , Células Cultivadas , Estudos de Coortes , Feminino , Humanos , Masculino , Mutação/genética , Podócitos/metabolismo , Rim Policístico Autossômico Dominante/fisiopatologia , Cuidados Pré-Operatórios , Prognóstico , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Sequenciamento do ExomaRESUMO
Increased osteoclastogenesis is responsible for osteolysis, which is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α-induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating the expression of microRNAs (miRNAs) important for this process. Using high-throughput sequencing of miRNAs, we obtained the first, to our knowledge, genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow-derived macrophages/osteoclast precursors during inflammatory osteoclastogenesis. Furthermore, we identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J-deficient cells. Complementary loss- and gain-of-function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and B lymphocyte-induced maturation protein-1. Moreover, we identified that direct miR-182 targets, Foxo3 and Maml1, play important inhibitory roles in TNF-α-mediated osteoclastogenesis. Thus, RBP-J-regulated miR-182 promotes TNF-α-induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α-induced osteoclastogenesis. Our results provide a novel miRNA-mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J-miR-182-Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone resorption.
Assuntos
Regulação da Expressão Gênica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , MicroRNAs/genética , Osteoclastos/metabolismo , Osteogênese , Fator de Necrose Tumoral alfa/metabolismo , Animais , Reabsorção Óssea , Regulação para Baixo , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Inflamação , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , MicroRNAs/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Current strategies to improve graft outcome following kidney transplantation consider information at the human leukocyte antigen (HLA) loci. Cell surface antigens, in addition to HLA, may serve as the stimuli as well as the targets for the anti-allograft immune response and influence long-term graft outcomes. We therefore performed exome sequencing of DNA from kidney graft recipients and their living donors and estimated all possible cell surface antigens mismatches for a given donor/recipient pair by computing the number of amino acid mismatches in trans-membrane proteins. We designated this tally as the allogenomics mismatch score (AMS). We examined the association between the AMS and post-transplant estimated glomerular filtration rate (eGFR) using mixed models, considering transplants from three independent cohorts (a total of 53 donor-recipient pairs, 106 exomes, and 239 eGFR measurements). We found that the AMS has a significant effect on eGFR (mixed model, effect size across the entire range of the score: -19.4 [-37.7, -1.1], P = 0.0042, χ2 = 8.1919, d.f. = 1) that is independent of the HLA-A, B, DR matching, donor age, and time post-transplantation. The AMS effect is consistent across the three independent cohorts studied and similar to the strong effect size of donor age. Taken together, these results show that the AMS, a novel tool to quantify amino acid mismatches in trans-membrane proteins in individual donor/recipient pair, is a strong, robust predictor of long-term graft function in kidney transplant recipients.
RESUMO
Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA, into oocytes in somatic cell nuclear transfer embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity.
Assuntos
Núcleo Celular/metabolismo , Reprogramação Celular , Regulação da Expressão Gênica no Desenvolvimento , Histonas/química , Oócitos/citologia , Animais , Cromatina/metabolismo , Citoplasma/metabolismo , Feminino , Camundongos , Técnicas de Transferência Nuclear , Oócitos/metabolismo , RNA Interferente Pequeno/metabolismo , Análise de Sequência de RNARESUMO
UNLABELLED: Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. KEY POINTS: Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.