RESUMEN
ERK1/2 (extracellular signal-regulated kinases 1 and 2) regulate the activity of various transcription factors that contribute to asthma pathogenesis. Although an attractive drug target, broadly inhibiting ERK1/2 is challenging because of unwanted cellular toxicities. We have identified small molecule inhibitors with a benzenesulfonate scaffold that selectively inhibit ERK1/2-mediated activation of AP-1 (activator protein-1). Herein, we describe the findings of targeting ERK1/2-mediated substrate-specific signaling with the small molecule inhibitor SF-3-030 in a murine model of house dust mite (HDM)-induced asthma. In 8- to 10-week-old BALB/c mice, allergic asthma was established by repeated intranasal HDM (25 µg/mouse) instillation for 3 weeks (5 days/week). A subgroup of mice was prophylactically dosed with 10 mg/kg SF-3-030/DMSO intranasally 30 minutes before the HDM challenge. Following the dosing schedule, mice were evaluated for alterations in airway mechanics, inflammation, and markers of airway remodeling. SF-3-030 treatment significantly attenuated HDM-induced elevation of distinct inflammatory cell types and cytokine concentrations in BAL and IgE concentrations in the lungs. Histopathological analysis of lung tissue sections revealed diminished HDM-induced pleocellular peribronchial inflammation, mucus cell metaplasia, collagen accumulation, thickening of airway smooth muscle mass, and expression of markers of cell proliferation (Ki-67 and cyclin D1) in mice treated with SF-3-030. Furthermore, SF-3-030 treatment attenuated HDM-induced airway hyperresponsiveness in mice. Finally, mechanistic studies using transcriptome and proteome analyses suggest inhibition of HDM-induced genes involved in inflammation, cell proliferation, and tissue remodeling by SF-3-030. These preclinical findings demonstrate that function-selective inhibition of ERK1/2 signaling mitigates multiple features of asthma in a murine model.
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Asma , Animales , Ratones , Asma/metabolismo , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inflamación/metabolismo , Pulmón/patología , Ratones Endogámicos BALB C , PyroglyphidaeRESUMEN
Interferon regulatory factor 8 (IRF8), a myeloid lineage transcription factor, emerges as an essential regulator for microglial activation. However, the precise role of IRF8 during Japanese encephalitis virus (JEV) infection in the brain remains elusive. Here, we report that JEV infection enhances IRF8 expression in the infected mouse brain. Comparative transcriptional profiling of whole-brain RNA analysis and validation by quantitative reverse transcription-PCR (qRT-PCR) reveals an impaired interferon gamma (IFN-γ) and related gene expression in Irf8 knockout (Irf8-/-)-infected mice. Further, Ifnγ knockout (Ifnγ-/-) mice exhibit a reduced level of Irf8. Both Ifnγ-/- and Irf8-/- mice exhibit significantly reduced levels of activated (CD11b+ CD45hi, CD11b+ CD45lo, Cd68, and CD86) and infiltrating immune cells (Ly6C+, CD4, and CD8) in the infected brain compared to those of wild-type (WT) mice. However, a higher level of granulocyte cell (Ly6G+) infiltration is evident in Irf8-/- mice as well as the increased concentration of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP1) levels in the brain. Interestingly, neither the Irf8-/- nor the Ifnγ-/- conferred protection against lethal JEV challenge to mice and exhibit augmentation in JEV replication in the brain. The gain of function of Irf8 by overexpressing functional IRF8 in an IRF8-deficient cell line attenuates viral replication and enhances IFN-γ production. Overall, we summarize that in the murine model of JEV encephalitis, IRF8 modulation affects JEV replication. We also show that lack of Irf8 affects immune cell abundance in circulation and the infected brain, leading to a reduction in IFN-γ level and increased viral load in the brain. IMPORTANCE Microglial cells, the resident macrophages in the brain, play a vital role in Japanese encephalitis virus (JEV) pathogenesis. The deregulated activity of microglia can be lethal for the brain. Therefore, it is crucial to understand the regulators that drive microglia phenotype changes and induce inflammation in the brain. Interferon regulatory factor 8 (IRF8) is a myeloid lineage transcription factor involved in microglial activation. However, the impact of IRF8 modulation on JEV replication remains elusive. Moreover, the pathways regulated by IRF8 to initiate and amplify pathological neuroinflammation are not well understood. Here, we demonstrated the effect of IRF8 modulation on JEV replication, microglial activation, and immune cells infiltration in the brain.
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Encéfalo/virología , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/inmunología , Factores Reguladores del Interferón/genética , Interferón gamma/inmunología , Replicación Viral/inmunología , Animales , Encéfalo/inmunología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Femenino , Regulación de la Expresión Génica/inmunología , Factores Reguladores del Interferón/inmunología , Interferón gamma/genética , Masculino , Ratones , Ratones Noqueados , Microglía/inmunología , Microglía/fisiología , Microglía/virología , Transducción de SeñalRESUMEN
Biological sex influences inflammatory response, as there is a greater incidence of acute inflammation in men and chronic inflammation in women. Here, we report that acute inflammation is attenuated by X-inactive specific transcript (Xist), a female cell-specific nuclear long noncoding RNA crucial for X-chromosome inactivation. Lipopolysaccharide-mediated acute inflammation increased Xist levels in the cytoplasm of female mouse J774A.1 macrophages and human AML193 monocytes. In both cell types, cytoplasmic Xist colocalizes with the p65 subunit of NF-κB. This interaction was associated with reduced NF-κB nuclear migration, suggesting a novel mechanism to suppress acute inflammation. Further supporting this hypothesis, expression of 5' XIST in male cells significantly reduced IL-6 and NF-κB activity. Adoptive transfer of male splenocytes expressing Xist reduced acute paw swelling in male mice indicating that Xist can have a protective anti-inflammatory effect. These findings show that XIST has functions beyond X chromosome inactivation and suggest that XIST can contribute to sex-specific differences underlying inflammatory response by attenuating acute inflammation in women.
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Inflamación/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Células Cultivadas , Citoplasma/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/patología , Inflamación/prevención & control , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Monocitos/metabolismo , FN-kappa B/metabolismo , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Factores Sexuales , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND: Preterm infants with bronchopulmonary dysplasia (BPD) have lifelong increased risk of respiratory morbidities associated with environmental pathogen exposure and underlying mechanisms are poorly understood. The resident immune cells of the lung play vital roles in host defense. However, the effect of perinatal events associated with BPD on pulmonary-specific immune cells is not well understood. METHODS: We used a double-hit model of BPD induced by prenatal chorioamnionitis followed by postnatal hyperoxia, and performed a global transcriptome analysis of all resident pulmonary immune cells. RESULTS: We show significant up-regulation of genes involved in chemokine-mediated signaling and immune cell chemotaxis, and down-regulation of genes involved in multiple T lymphocyte functions. Multiple genes involved in T cell receptor signaling are downregulated and Cd8a gene expression remains downregulated at 2 months of age in spite of recovery in normoxia for 6 weeks. Furthermore, the proportion of CD8a+CD3+ pulmonary immune cells is decreased. CONCLUSIONS: Our study has highlighted that perinatal lung inflammation in a double-hit model of BPD results in short- and long-term dysregulation of genes associated with the pulmonary T cell receptor signaling pathway, which may contribute to increased environmental pathogen-associated respiratory morbidities seen in children and adults with BPD. IMPACT: In a translationally relevant double-hit model of BPD induced by chorioamnionitis and postnatal hyperoxia, we identified pulmonary immune cell-specific transcriptomic changes and showed that T cell receptor signaling genes are downregulated in short term and long term. This is the first comprehensive report delineating transcriptomic changes in resident immune cells of the lung in a translationally relevant double-hit model of BPD. Our study identifies novel resident pulmonary immune cell-specific targets for potential therapeutic modulation to improve short- and long-term respiratory health of preterm infants with BPD.
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Displasia Broncopulmonar/genética , Corioamnionitis/patología , Hiperoxia/complicaciones , Pulmón/inmunología , Transcriptoma , Animales , Displasia Broncopulmonar/etiología , Modelos Animales de Enfermedad , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVES: Systemic sclerosis (SSc) is characterised by severe fibroproliferative vasculopathy, fibrosis in skin and multiple internal organs, and humoral, cellular and innate immunity abnormalities. Vascular alterations are the earliest and most severe SSc manifestations, however, the mechanisms responsible have remained elusive. To investigate the molecular abnormalities involved in SSc-vasculopathy we examined global gene expression differences between highly purified lung microvascular endothelial cells (MVECs) from patients with SSc-interstitial lung disease (SSc-ILD) and normal lung MVECs. METHODS: MVECs were isolated from fresh transplanted lungs from patients with SSc-ILD. Sequential CD31 and CD102 immunopurification was performed to obtain highly purified CD31+/CD102+ lung MVECs. Global gene expression analysis was successfully performed in CD31+/CD102+ MVEC from two SSc-ILD patients and from two normal lungs. RT-PCR, Western blots, and indirect immunofluorescence validated the gene expression results. RESULTS: Numerous interferon-regulated genes (IRGs) including IFI44, IFI44L, IFI6, IFIH1, IFIT1, ISG-15, BST-2/Tetherin, and RSAD2/Viperin, genes encoding innate immunity antiviral responses (OAS1, OAS2, OAS3, OASL) and antiviral MX1 and MX2 proteins, and mesenchymal cell-specific genes were significantly overexpressed in CD31+/CD102+ SSc-ILD lung MVECs. CONCLUSIONS: Highly purified CD31+/CD102+ MVECs from lungs from SSc patients with end stage SSc-ILD displayed remarkable overexpression of numerous IRGs and of genes encoding antiviral innate immune response and antiviral proteins. These observations suggest that interferon-induced and antiviral response proteins may participate in the pathogenesis of SSc vasculopathy and SSc-ILD. The CD31+/CD102+ lung MVECs from SSc-ILD also showed elevated expression of mesenchymal cell-specific genes confirming the presence of endothelial to mesenchymal transition in SSc-ILD.
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Factores de Restricción Antivirales/genética , Interferones , Enfermedades Pulmonares Intersticiales , Esclerodermia Sistémica , Células Endoteliales , Humanos , Pulmón , Enfermedades Pulmonares Intersticiales/genética , Esclerodermia Sistémica/genéticaRESUMEN
CACX is one of the most common cancer affecting women world-wide. Here, expression microarray analysis revealed 8 over-expressed transcribed pseudogenes (GBP1P1, HLA-DRB6, HLA-H, SLC6A10P, NAPSB, KRT16P2, PTTG3P and RNF126P1), down-regulated 7 lincRNAs (H19, MIR100HG, MEG3, DIO3OS, HOXA11-AS, CD27-AS1 and EPB41L4A-AS) and 6 snoRNAs (SNORD97, SNORD3A, SNORD3C, SNORD3D, SNORA12 and SCARNA9) as DEncGs (log2 fold-changeâ¯≥⯱1.0) in CACX. Consequently, down-regulation of lincRNA MEG3 and over-expression of pseudogenes, GBP1P1 and PTTG3P in the microarray analysis were found concordant with the real-time quantitative PCR results upon validation. Then, Ingenuity® Pathway analysis (IPA®) analysis with deregulated DEncGs identified functionally important gene, H19. Further, validation (nâ¯=â¯52) of expression confirmed frequent downregulation of H19 with significant association with its deletion (LOH) and promoter methylation (nâ¯=â¯128) in CACX. Moreover, clinicopathological analysis found Indian CACX patients (nâ¯=â¯26) with alterations of H19 by deletion or, promoter methylation with concomitant low expression have poor prognosis.
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Carcinoma/genética , ARN Largo no Codificante/genética , Neoplasias del Cuello Uterino/genética , Carcinoma/metabolismo , Carcinoma/mortalidad , Carcinoma/patología , Línea Celular Tumoral , Metilación de ADN , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , India , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Regiones Promotoras Genéticas , Seudogenes , ARN Largo no Codificante/metabolismo , ARN Nucleolar Pequeño/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/mortalidad , Neoplasias del Cuello Uterino/patologíaRESUMEN
Arsenic in drinking water is one of the major etiological factors in urinary bladder carcinoma (BlCa). Here, high-resolution CGH-SNP microarray analysis in arsenic accumulated BlCa tissues showed significant (p < 0.05) association of chromosomal alterations with high arsenic (≥112 ng/g) accumulation, further corroborated by high γH2AX nuclear expression. Cytobands 5q11-35, 9p24.3-21.5, 18q11.1-25, etc. showed deletion, whereas 12q was amplified in high arsenic samples (AsH). Consecutively, IPA® found FA-BRCA pathway to be exclusively altered in AsH group. Validation of several key regulatory genes (RAD50, BRIP1, UIMC1, FANCD2, BRCA2 and BRCA1) of the pathway, were performed in independent BlCa cases (n = 81). UIMC1, RAD50 and BRIP1 were differentially deleted and associated with poor survival of AsH samples. Moreover, reduced nuclear expression with diffused cytoplasmic expression of FANCD2 was higher in AsH samples. Collectively, frequent deregulation of RAD50, UIMC1 and BRIP1 may result in reduced nuclear translocation of FANCD2, which may cause more chromosomal aberrations among AsH samples.
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Arsénico/toxicidad , Carcinoma/genética , Neoplasias de la Vejiga Urinaria/genética , Arsénico/metabolismo , Carcinoma/etiología , Carcinoma/metabolismo , Carcinoma/mortalidad , Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN , Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Perfilación de la Expresión Génica , Genes BRCA1 , Genes BRCA2 , Genómica , Redes y Vías Metabólicas , Repeticiones de Microsatélite , Transducción de Señal , Neoplasias de la Vejiga Urinaria/etiología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/mortalidadRESUMEN
Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.
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Caveolinas/antagonistas & inhibidores , Factor de Transcripción GATA6/metabolismo , Hipertrofia/patología , Músculo Liso/patología , FN-kappa B/metabolismo , Transcripción Genética , Obstrucción del Cuello de la Vejiga Urinaria/complicaciones , Anciano , Animales , Biomarcadores/análisis , Caveolinas/genética , Caveolinas/metabolismo , Factor de Transcripción GATA6/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Hipertrofia/etiología , Hipertrofia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Contracción Muscular , Músculo Liso/metabolismo , FN-kappa B/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Obstrucción del Cuello de la Vejiga Urinaria/cirugíaRESUMEN
BACKGROUND: CSCC is one of the most common cancer affecting women globally. Though it is caused by the infection of hrHPV but long latency period for malignant outcome in only a subset of hrHPV infected women indicates involvement of additional alterations, primarily CNVs. Here, we showed how CNVs played a crucial role in development of advanced tumors (stage III/IV) in Indian patients. METHODS: Initially, high-resolution CGH-SNP microarray analysis pointed out frequent CNVs followed by significantly altered genes. After comparison with TCGA dataset, expressions of the genes were checked in three CSCC datasets to identify key genes followed by Ingenuity® Pathway analysis. Then node effect property analysis was applied on the constructed PPI network to rank the key proteins. Finally, validations in independent samples were performed. RESULTS: For the first time, frequent chromosomal amplifications at 3q13.13-3q29, 1p36.11-1p31.1, 1q21.1-1q44 and 5p15.33-5p12 followed by common deletions at 11q14.1-11q25, 2q34-2q37.3, 4p16.3-4p12 and 13q13.3-13q14.3 were identified in Indian CSCC patients. Integrative analysis found 78 key genes including several novel ones, which were mostly associated with 'Cancer' and may regulate DNA repair and metabolic pathways. Analysis showed PARP1 and ATR were among the top ranking protein interactors. CONCLUSIONS: Frequent amplification and over-expression of ATR and PARP1 were further confirmed in cervical lesions, indicating their association with poor prognosis of advanced CSCC patients. GENERAL SIGNIFICANCE: Our novel approach identified precise CNVs along with several novel genes within these loci and showed that PARP1 and ATR, having biologically significant interactions, may be involved in development of advanced CSCC.
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Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Redes Reguladoras de Genes , Genómica , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Línea Celular Tumoral , Cromosomas Humanos/genética , Variaciones en el Número de Copia de ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Humanos , Estadificación de Neoplasias , Polimorfismo de Nucleótido Simple/genética , Mapas de Interacción de Proteínas/genética , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis. We recently found that focal adhesion kinase 1 (FAK1) is upregulated and phosphorylated (active) in IBC. In this study, we investigated the effect of CEP-37440, a dual inhibitor of FAK1 and anaplastic lymphoma kinase (ALK), using human IBC cell lines and preclinical models of IBC. METHODS: Cell proliferation assays were performed in the presence of several concentrations of CEP-37440 using IBC and triple-negative breast cancer non-IBC cell lines. In vitro, we studied the expression of total FAK1, phospho-FAK1 (Tyr 397), total ALK and phospho-ALK (Tyr 1604). In vivo, we tested CEP-37440 using FC-IBC02, SUM149, and SUM190 IBC xenograft mouse models. RESULTS: CEP-37440 at low concentration decreased the proliferation of the IBC cell lines FC-IBC02, SUM190, and KPL4, while not affecting the proliferation of normal breast epithelial cells. At higher concentration, CEP-37440 was also able to inhibit the proliferation of the IBC cell line MDA-IBC03 and the triple-negative non-IBC cell lines MDA-MB-231 and MDA-MB-468; the IBC cell line SUM149 showed a slight response to the drug. CEP-37440 decreased the cell proliferation of FC-IBC02, SUM190, and KPL4 by blocking the autophosphorylation kinase activity of FAK1 (Tyr 397). None of the cells evaluated expressed ALK. In vivo, after 7 weeks of CEP-37440 treatment, the SUM190, FC-IBC02, and SUM149 breast tumor xenografts were smaller in mice treated with 55 mg/kg bid CEP-37440 compared to the controls; the tumor growth inhibition (TGI) was 79.7 %, 33 %, and 23 %, respectively. None of the FC-IBC02 breast xenografts mice treated with CEP-37440 developed brain metastasis while 20 % of the mice in the control group developed brain metastasis. Expression array analyses in FC-IBC02 cells showed that CEP-37440 affects the expression of genes related to apoptosis, interferon signaling, and cytokines. CONCLUSIONS: CEP-37440 is effective against some IBC cells that express phospho-FAK1 (Tyr 397), and its antiproliferative activity is related to its ability to decrease phospho-FAK1. Our results suggest that combinational therapies could be more effective than using CEP-37440 as a single agent.
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Benzamidas/administración & dosificación , Benzocicloheptenos/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Neoplasias Inflamatorias de la Mama/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Quinasa de Linfoma Anaplásico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Proteína-Tirosina Quinasas de Adhesión Focal/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Inflamatorias de la Mama/genética , Neoplasias Inflamatorias de la Mama/patología , Ratones , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The presence of immunoglobulin oligoclonal bands in the cerebrospinal fluid of Multiple Sclerosis (MS) patients supports the hypothesis of an infectious etiology, although the antigenic targets remain elusive. Neurotropic mouse hepatitis virus (MHV) infection in mice provides a useful tool for studying mechanisms of demyelination in a virus-induced experimental model of MS. This study uses Affymetrix microarray analysis to compare differential spinal cord mRNA levels between mice infected with demyelinating and non-demyelinating strains of MHV to identify host immune genes expressed in this demyelinating disease model. The study reveals that during the acute stage of infection, both strains induce inflammatory innate immune response genes, whereas upregulation of several immunoglobulin genes during chronic stage infection is unique to infection with the demyelinating strain. Results suggest that the demyelinating strain induced an innate-immune response during acute infection that may promote switching of Ig isotype genes during chronic infection, potentially playing a role in antibody-mediated progressive demyelination even after viral clearance.
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Inmunidad Adaptativa/genética , Infecciones por Coronavirus/genética , Enfermedades Desmielinizantes/genética , Regulación de la Expresión Génica/genética , Inmunidad Innata/genética , Animales , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/virología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunohistoquímica , Ratones Endogámicos C57BL , Virus de la Hepatitis Murina/inmunología , Virus de la Hepatitis Murina/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/inmunología , Médula Espinal/metabolismo , Médula Espinal/virologíaRESUMEN
A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A2 analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence.
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Envejecimiento/metabolismo , Canal Anal/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Músculo Liso/metabolismo , Envejecimiento/genética , Animales , Perfilación de la Expresión Génica , Factor 4 Similar a Kruppel , MicroARNs/genética , Contracción Muscular/fisiología , Ratas , Ratas Endogámicas F344 , Transducción de Señal/fisiologíaRESUMEN
During myocardial ischemia, upregulation of the hedgehog (Hh) pathway promotes neovascularization and increases cardiomyocyte survival. The canonical Hh pathway activates a transcriptional program through the Gli family of transcription factors by derepression of the seven-transmembrane protein smoothened (Smo). The mechanisms linking Smo to Gli are complex and, in some cell types, involve coupling of Smo to Gi proteins. In the present study, we investigated, for the first time, the transcriptional response of cardiomyocytes to sonic hedgehog (Shh) and the role of Gi protein utilization. Our results show that Shh strongly activates Gli1 expression by quantitative PCR in a Smo-dependent manner in neonatal rat ventricular cardiomyocytes. Microarray analysis of gene expression changes elicited by Shh and sensitive to a Smo inhibitor identified a small subset of 37 cardiomyocyte-specific genes regulated by Shh, including some in the PKA and purinergic signaling pathways. In addition, neonatal rat ventricular cardiomyocytes infected with an adenovirus encoding GiCT, a peptide that impairs receptor-Gi protein coupling, showed reduced activation of Hh targets. In vitro data were confirmed in transgenic mice with cardiomyocyte-inducible GiCT expression. Transgenic GiCT mice showed specific reduction of Gli1 expression in the heart under basal conditions and failed to upregulate the Hh pathway upon ischemia and reperfusion injury, unlike their littermate controls. This study characterizes, for the first time, the transcriptional response of cardiomyocytes to Shh and establishes a critical role for Smo coupling to Gi in Hh signaling in the normal and ischemic myocardium.
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Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Hedgehog/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Ratones Transgénicos , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Receptor Smoothened , Proteína con Dedos de Zinc GLI1RESUMEN
Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent "mark" on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these "cured" neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that can persist or progress in the absence of sustained viral antigen.
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Encéfalo/virología , Neuronas/fisiología , Virus de la Rabia/inmunología , Rabia/inmunología , Animales , Encéfalo/inmunología , Encéfalo/patología , Supervivencia Celular , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Integrasas/genética , Ratones , Neuronas/inmunología , Neuronas/virología , Rabia/genética , Rabia/patología , Rabia/virología , Virus de la Rabia/patogenicidad , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
Background Understanding the genetic basis for the molecular classification of sinonasal undifferentiated carcinoma (SNUC) based on SMARCB1 may improve our understating regarding the nature of the disease. The objective of the study was to compare the genetic profile of SMARCB1-retained (SR-SNUC) and SMARCB1-deficient SNUC (SD-SNUC). Methods Formalin-fixed, paraffin-embedded tissue from treatment-naive patients with SNUC were selected. Three cases of SR-SNUC, four cases of SD-SNUC, and four samples of nontumor tissue (control samples) were selected. Ribonucleic acid (RNA) sequencing was performed. Results SR-SNUC had a higher number of variants (1 variant for every 15,000 bases) compared with SD-SNUC (1 variant every 29,000 bases). The ratio of missense to silent mutation ratio was higher for SR-SNUC (0.8) as compared with SD-SNUC (0.7). Approximately 1,500 genes were differentially expressed between SR-SNUC and SD-SNUC. The genes that had a higher expression in SR-SNUC included TPD52L1, B3GNT3, GFY, TJP3, ELL3, CYP4F3, ALDH3B2, CKMT1B, VIPR1, SLC7A5, PPP2R2C, UPK3B, MUC1, ELF5, STY7, and H2AC14. The gene that had a higher expression in SD-SNUC was ZFHX4. Most of these genes were related to either protein translation or immune regulation. The most common ( n = 3, 75%) mechanisms of loss of SMARCB1 gene in SD-SNUC was loss of heterozygosity. Conclusion RNA sequencing is a viable and informative approach for genomic profiling of archival SNUC samples. Both SR-SNUC and SD-SNUC were noted to have distinct genetic profiles underlying the molecular classification of these diseases.
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Methadone maintenance treatment for opioid dependent mothers is standard of care. Infants of methadone maintained opioid dependent (MMOD) mothers have better outcomes compared to infants of opioid dependent mothers without treatment. However, when compared to non-exposed infants, infants of MMOD mothers are associated with worse outcomes. We conducted a pilot study to examine genome wide differential DNA methylation using cord blood samples from sixteen term and near-term infants of MMOD and opioid naïve mothers, excluding Infants with chorioamnionitis. A total of 152 differentially methylated loci were identified at a difference > + 2, < - 2 and p-value < 0.05. There were 90 hypermethylated loci (59 annotated genes) and 62 hypomethylated loci (38 annotated genes) observed. The hypermethylated and hypomethylated DNA changes involved multiple genes, pathways and networks that may explain some of the changes seen in infants of MMOD mothers. Top hypermethylated and hypomethylated genes involved areas of cell growth, neurodevelopment, vision and xenobiotic metabolism functions. Our data may explain the role of key pathways and genes relevant to neonatal outcomes seen from methadone exposure in pregnancy. Functional studies on the identified pathways and genes could lead to improved understanding of the mechanisms and identify areas for intervention.
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Metilación de ADN , Sangre Fetal , Metadona , Trastornos Relacionados con Opioides , Humanos , Femenino , Metadona/uso terapéutico , Sangre Fetal/metabolismo , Embarazo , Trastornos Relacionados con Opioides/genética , Recién Nacido , Adulto , Proyectos Piloto , Tratamiento de Sustitución de Opiáceos , Complicaciones del Embarazo/genética , Complicaciones del Embarazo/tratamiento farmacológico , Masculino , Analgésicos Opioides/uso terapéutico , MadresRESUMEN
Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, ß-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6-4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10-3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.
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Regulación Neoplásica de la Expresión Génica , Neoplasias Inflamatorias de la Mama/genética , Neoplasias Inflamatorias de la Mama/patología , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/genética , Quinasa de Linfoma Anaplásico , Animales , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Antígenos CD4/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Cromosomas Humanos Par 8 , Proteínas de Unión al ADN/genética , Transición Epitelial-Mesenquimal , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Genes myc , Humanos , Neoplasias Inflamatorias de la Mama/metabolismo , Pérdida de Heterocigocidad , Neoplasias Pulmonares/secundario , Ratones , Ratones SCID , Terapia Molecular Dirigida , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Notch3 , Receptores Notch/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Background: The global pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). About 18.4% of total Covid-19 cases were reported in children. Even though vertical transmission from mother to infant is likely to occur at a low rate, exposure to COVID-19 during fetal life may alter DNA methylation patterns with potential long-term effects. Objective: To determine if COVID-19 infection during pregnancy alters the DNA methylation patterns in umbilical cord blood cells from term infants and to identify potential pathways and genes affected by exposure to COVID-19 infection. Methods: Umbilical cord blood was collected from 8 infants exposed to COVID-19 during pregnancy and 8 control infants with no COVID-19 exposure. Genomic DNA was isolated from umbilical cord blood cells and genome-wide DNA methylation was performed using Illumina Methylation EPIC Array. Results: 119 differentially methylated loci were identified at the FDR level of 0.20 (64 hypermethylated loci and 55 hypomethylated loci) in umbilical cord blood cells of COVID-19 exposed neonates compared to the control group. Important canonical pathways identified by Ingenuity Pathway Analysis (IPA) were related to stress response (corticotropin releasing hormone signaling, glucocorticoid receptor signaling, and oxytocin in brain signaling pathway), and cardiovascular disease and development (nitric oxide signaling in the cardiovascular system, apelin cardiomyocyte signaling pathways, factors promoting cardiogenesis, and renin-angiotensin signaling). The genes affected by the differential methylations were associated with cardiac, renal, hepatic, neurological diseases, developmental and immunological disorders. Conclusions: COVID-19 induces differential DNA methylation in umbilical cord blood cells. The differentially methylated genes may contribute to hepatic, renal, cardiac, developmental and immunological disorders in offspring born to mothers with COVID-19 infection during pregnancy, and their developmental regulation.
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Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFb activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFb kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.