RESUMEN
Human cytomegalovirus (HCMV) is a large DNA herpesvirus that is highly prevalent in the human population. HCMV can result in severe direct and indirect pathologies under immunosuppressed conditions and is the leading cause of birth defects related to infectious disease. Currently, the effect of HCMV infection on host cell metabolism as an increase in glycolysis during infection has been defined. We have observed that oxidative phosphorylation is also increased. We have identified morphological and functional changes to host mitochondria during HCMV infection. The mitochondrial network undergoes fission events after HCMV infection. Interestingly, the network does not undergo fusion. At the same time, mitochondrial mass and membrane potential increase. The electron transport chain (ETC) functions at an elevated rate, resulting in the release of increased reactive oxygen species. Surprisingly, despite the stress applied to the host mitochondria, the network is capable of responding to and meeting the increased bioenergetic and biosynthetic demands placed on it. When mitochondrial DNA is depleted from the cells, we observed severe impairment of viral replication. Mitochondrial DNA encodes many of the ETC components. These findings suggest that the host cell ETC is essential to HCMV replication. Our studies suggest the host cell mitochondria may be a therapeutic target.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus present in up to 85% of some populations. Like all herpesviruses, HCMV infection is for life. No vaccine is currently available, neutralizing antibody therapies are ineffective, and current antivirals have limited long-term efficacy due to side effects and potential for viral mutation and resistance. The significance of this research is in understanding how HCMV manipulates the host mitochondria to support bioenergetic and biosynthetic requirements for replication. Despite a large genome, HCMV relies exclusively on host cells for metabolic functions. By understanding the dependency of HCMV on the mitochondria, we could exploit these requirements and develop novel antivirals.
Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Citomegalovirus/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Línea Celular , Infecciones por Citomegalovirus/patología , Humanos , Mitocondrias/patologíaRESUMEN
UNLABELLED: In the oral epithelium, peripheral stores of Epstein-Barr virus (EBV) are transmitted from infiltrating B cells to epithelial cells. Once the virus is transmitted to epithelial cells, the highly permissive nature of this cell type for lytic replication allows virus amplification and exchange to other hosts. Since the initial transfer of EBV from B cells to epithelial cells requires transitioning of the B-cell to a state that induces virus reactivation, we hypothesized that there might be epithelium-specific signals that allow the infiltrating B cells to sense the appropriate environment to initiate reactivation and begin this exchange process. We previously found that the epithelium-specific miR-200 family of microRNAs promotes EBV lytic replication. Here we show that there are high levels of miR-200 family members in oral and tonsillar epithelia and in saliva. Analysis of cultured oral epithelial cells (OKF6) showed that they actively secrete membrane vesicles (exosomes) that are enriched with miR-200 family members. Coculturing of EBV-positive B cells with OKF6 cells induced viral reactivation. Further, treatment of EBV-positive B cells with OKF6 cell-derived membrane vesicles promoted reactivation. Using a cell system that does not naturally express miR-200 family members, we found that enforced expression of a miR-200 family member produced membrane vesicles that were able to induce the lytic cascade in EBV-positive B cells. We propose that membrane vesicles secreted by oral and tonsillar epithelial cells may serve as a tissue-specific environmental cue that initiates reactivation in B cells, promoting the transfer of virus from peripheral B-cell stores to the oral epithelium to facilitate virus amplification and exchange to other hosts. IMPORTANCE: Epstein-Barr virus (EBV) is an important human pathogen that is causally associated with several lymphomas and carcinomas. The switch from latency to the lytic cycle is critical for successful host infection and for EBV pathogenesis. Although the EBV lytic cycle can be triggered by certain agents in vitro, the mechanisms that signal reactivation in vivo are poorly understood. We previously reported that endogenously expressed miR-200 family members likely play a role in facilitating the lytic tendencies of EBV in epithelial cells. Here we show that membrane vesicles secreted from oral epithelial cells contain miR-200 family members and that they can be transmitted to proximal EBV-positive B cells, where they trigger reactivation. We propose that this intercellular communication pathway may serve as a sensor mechanism for infiltrating B cells to recognize an appropriate environment to initiate reactivation, thereby allowing the exchange of virus to the oral epithelium.
Asunto(s)
Linfocitos B/virología , Células Epiteliales/virología , Vesículas Extracelulares/virología , Herpesvirus Humano 4/fisiología , MicroARNs/genética , Mucosa Bucal/virología , Activación Viral/fisiología , Replicación Viral , Células HEK293 , Interacciones Huésped-Patógeno/fisiología , Humanos , Microscopía Electrónica de Transmisión , Mucosa Bucal/citología , Tonsila Palatina/citología , Tonsila Palatina/virología , Saliva/virologíaRESUMEN
Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC - high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.
Asunto(s)
Bases de Datos de Ácidos Nucleicos , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Inmunoterapia , Neoplasias Gástricas , Microambiente Tumoral , Adulto , Anciano , Anciano de 80 o más Años , Infecciones por Virus de Epstein-Barr/epidemiología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/terapia , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Regulación Viral de la Expresión Génica/genética , Regulación Viral de la Expresión Génica/inmunología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Neoplásico/inmunología , ARN Viral/biosíntesis , ARN Viral/genética , ARN Viral/inmunología , Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/genética , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapia , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Adipose-derived stromal/stem cells (ASCs) have anti-inflammatory as well as immunosuppressive activities and are currently the focus of clinical trials for a number of inflammatory diseases. Acute lung injury (ALI) is an inflammatory condition of the lung for which standard treatment is mainly supportive due to lack of effective therapies. Our recent studies have demonstrated the ability of both human ASCs (hASCs) and mouse ASCs (mASCs) to attenuate lung damage and inflammation in a rodent model of lipopolysaccharide-induced ALI, suggesting that ASCs may also be beneficial in treating ALI. To better understand how ASCs may act in ALI and to elucidate the mechanism(s) involved in ASC modulation of lung inflammation, gene expression analysis was performed in ASC-treated (hASCs or mASCs) and control sham-treated lungs. The results revealed a dramatic difference between the expression of anti-inflammatory molecules by hASCs and mASCs. These data show that the beneficial effects of hASCs and mASCs in ALI may result from the production of different paracrine factors. Interleukin 6 (IL-6) expression in the mASC-treated lungs was significantly elevated as compared to sham-treated controls 20 hours after delivery of the cells by oropharyngeal aspiration. Knockdown of IL-6 expression in mASCs by RNA interference abrogated most of their therapeutic effects, suggesting that the anti-inflammatory properties of mASCs in ALI are explained, at least in part, by activation of IL-6 secretion.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/terapia , Tejido Adiposo/citología , Interleucina-6/metabolismo , Trasplante de Células Madre , Células Madre/citología , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Albúminas/metabolismo , Animales , Antiinflamatorios/metabolismo , Líquido del Lavado Bronquioalveolar , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Factor Inhibidor de Leucemia/metabolismo , Lipopolisacáridos , Pulmón/patología , Ratones Endogámicos C57BL , Células del EstromaRESUMEN
Recent findings demonstrate that inhaled cigarette smoke, the predominant lung carcinogen, elicits a T helper 17 (Th17) inflammatory phenotype. Interleukin-17A (IL-17), the hallmark cytokine of Th17 inflammation, displays pro- and antitumorigenic properties in a manner that varies according to tumor type and assay system. To investigate the role of IL-17 in lung tumor growth, we used an autochthonous tumor model (K-Ras(LA1) mice) with lung delivery of a recombinant adenovirus that expresses IL-17A. Virus-mediated expression of IL-17A in K-Ras(LA1) mice at 8-10 wk of age doubled lung tumor growth in 3 wk relative to littermates that received a green fluorescent protein-expressing control adenovirus. IL-17 induced matrix metalloproteinase-9 (MMP-9) expression in vivo and in vitro. In accord with this finding, selective and specific inhibitors of MMP-9 repressed the increased motility and invasiveness of IL-17-treated lung tumor cells in culture. Knockdown or mutation of p53 promoted the motility of murine lung tumor cells and abrogated the promigratory role of IL-17. Coexpression of siRNA-resistant wild-type, but not mutant, human p53 rescued both IL-17-mediated migration and MMP-9 mRNA induction in p53 knockdown lung tumor cells. IL-17 increased MMP-9 mRNA stability by reducing interaction with the mRNA destabilizing serine/arginine-rich splicing factor 1 (SRSF1). Taken together, our results indicate that IL-17 stimulates lung tumor growth and regulates MMP-9 mRNA levels in a p53- and SRSF1-dependent manner.
Asunto(s)
Movimiento Celular , Interleucina-17/biosíntesis , Neoplasias Pulmonares/metabolismo , Animales , Estabilidad de Enzimas/genética , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-17/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Transgénicos , Invasividad Neoplásica , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factores de Empalme de ARN , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that currently infects a large percentage of the world population. Although usually asymptomatic in healthy individuals, HCMV infection during pregnancy may cause spontaneous abortions, premature delivery, or permanent neurological disabilities in infants infected in utero. During infection, the virus exerts control over a multitude of host signaling pathways. Wnt/ß-catenin signaling, an essential pathway involved in cell cycle control, differentiation, embryonic development, placentation and metastasis, is frequently dysregulated by viruses. How HCMV infection affects this critical pathway is not currently known. In this study, we demonstrate that HCMV dysregulates Wnt/ß-catenin signaling in dermal fibroblasts and human placental extravillous trophoblasts. Infection inhibits Wnt-induced transcriptional activity of ß-catenin and expression of ß-catenin target genes in these cells. HCMV infection leads to ß-catenin protein accumulation in a discrete juxtanuclear region. Levels of ß-catenin in membrane-associated and cytosolic pools, as well as nuclear ß-catenin, are reduced after infection; while transcription of the ß-catenin gene is unchanged, suggesting enhanced degradation. Given the critical role of Wnt/ß-catenin signaling in cellular processes, these findings represent a novel and important mechanism whereby HCMV disrupts normal cellular function.
Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/metabolismo , Ciclo Celular , Diferenciación Celular , Línea Celular , Movimiento Celular , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Humanos , Transcripción Genética , Activación Transcripcional , Trofoblastos/metabolismo , Trofoblastos/virologíaRESUMEN
BACKGROUND: KSHV is a tumorigenic γ-herpesvirus that has been identified as the etiologic agent of Kaposi's sarcoma (KS), a multifocal highly vascularized neoplasm that is the most common malignancy associated with acquired immunodeficiency syndrome (AIDS). The virus encodes a constitutively active chemokine receptor homologue, vGPCR that possesses potent angiogenic and tumorigenic properties, and is critical for KSHV pathobiology. To date, a number of signaling pathways have been identified as key in mediating vGPCR oncogenic potential. FINDINGS: In this study, we identify a novel pathway, the Wnt/ß-catenin pathway, which is dysregulated by vGPCR expression in endothelial cells. Expression of vGPCR in endothelial cells enhances the nuclear accumulation of ß-catenin, that correlates with an increase in ß-catenin transcriptional activity. Activation of ß-catenin signaling by vGPCR is dependent on the PI3K/Akt pathway, as treatment of vGPCR-expressing cells with a pharmacological inhibitor of PI3K, leads to a decreased activation of a ß-catenin-driven reporter, a significant decrease in expression of ß-catenin target genes, and reduced endothelial tube formation. CONCLUSIONS: Given the critical role of Wnt/ß-catenin signaling in angiogenesis and tumorigenesis, the findings from this study suggest a novel mechanism in KSHV-induced malignancies.
Asunto(s)
Herpesvirus Humano 8/fisiología , Interacciones Huésped-Patógeno , Receptores Acoplados a Proteínas G/metabolismo , Vía de Señalización Wnt , Animales , Células Cultivadas , Células Endoteliales/virología , Humanos , Ratones DesnudosRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is a major food-borne pathogen that causes human disease ranging from diarrhea to life-threatening complications. Accumulating evidence demonstrates that the Western diet enhances the susceptibility to enteric infection in mice, but the effect of diet on EHEC colonization and the role of human gut microbiota remains unknown. Our research aimed to investigate the effects of a Standard versus a Western diet on EHEC colonization in the human in vitro Mucosal ARtificial COLon (M-ARCOL) and the associated changes in the gut microbiota composition and activities. After donor selection using simplified fecal batch experiments, two M-ARCOL bioreactors were inoculated with a human fecal sample (n = 4) and were run in parallel, one receiving a Standard diet, the other a Western diet and infected with EHEC O157:H7 strain EDL933. EHEC colonization was dependent on the donor and diet in the luminal samples, but was maintained in the mucosal compartment without elimination, suggesting a favorable niche for the pathogen, and may act as a reservoir. The Western diet also impacted the bacterial short-chain fatty acid and bile acid profiles, with a possible link between high butyrate concentrations and prolonged EHEC colonization. The work demonstrates the application of a complex in vitro model to provide insights into diet, microbiota, and pathogen interactions in the human gut.
Asunto(s)
Colon , Dieta Occidental , Escherichia coli Enterohemorrágica , Heces , Microbioma Gastrointestinal , Humanos , Microbioma Gastrointestinal/fisiología , Dieta Occidental/efectos adversos , Colon/microbiología , Heces/microbiología , Infecciones por Escherichia coli/microbiología , Mucosa Intestinal/microbiología , Mucosa Intestinal/metabolismo , Ácidos Grasos Volátiles/metabolismo , Ácidos y Sales Biliares/metabolismo , Escherichia coli O157RESUMEN
The Australian National Preventive Health Strategy 2021-2030 recommended the establishment of evidence-based frameworks to enable local public health services to identify strategies and interventions that deliver value for money. This study aimed to review the cost-effectiveness of preventive health strategies to inform the reorientation of local public health services towards preventive health interventions that are financially sustainable. Four electronic databases were searched for reviews published between 2005 and February 2022. Reviews that met the following criteria were included: population: human studies, any age or sex; concept 1: primary and/or secondary prevention interventions; concept 2: full economic evaluation; context: local public health services as the provider of concept 1. The search identified 472 articles; 26 were included. Focus health areas included mental health (n = 3 reviews), obesity (n = 1), type 2 diabetes (n = 3), dental caries (n = 2), public health (n = 4), chronic disease (n = 5), sexual health (n = 1), immunisation (n = 1), smoking cessation (n = 3), reducing alcohol (n = 1), and fractures (n = 2). Interventions that targeted obesity, type 2 diabetes, smoking cessation, and fractures were deemed cost-effective, however, more studies are needed, especially those that consider equity in priority populations.
Asunto(s)
Caries Dental , Diabetes Mellitus Tipo 2 , Fracturas Óseas , Humanos , Análisis Costo-Beneficio , Australia , Servicios Preventivos de Salud , Obesidad/prevención & controlRESUMEN
BACKGROUND: During the first trimester of pregnancy, a series of tightly regulated interactions govern the formation of a highly invasive population of fetal-derived extravillous cytotrophoblasts (EVT). Successful pregnancy is dependent on efficient invasion of the uterine wall and maternal spiral arteries by EVT. Dysregulated trophoblast invasion is associated with intrauterine growth restriction, birth defects, spontaneous abortion and preeclampsia. A number of soluble growth factors, cytokines, and chemokines modulate this process, fine-tuning the temporal and spatial aspects of cytotrophoblast invasion. In particular, the CXCL12/CXCR4 axis has been shown to specifically modulate cytotrophoblast differentiation, invasion, and survival throughout early pregnancy. Infection with human cytomegalovirus (HCMV) has been associated with impaired differentiation of cytotrophoblasts down the invasive pathway, specifically dysregulating the response to mitogens including epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In this study, the effect of HCMV infection on the CXCL12-mediated migration and invasion of the EVT cell line SGHPL-4 was investigated. RESULTS: Infection with HCMV significantly decreased secretion of CXCL12 by SGHPL-4 cells, and induced a striking perinuclear accumulation of the chemokine. HCMV infection significantly increased mRNA and total cell surface expression of the two known receptors for CXCL12: CXCR4 and CXCR7. Functionally, HCMV-infected SGHPL-4 cells were unable to migrate or invade in response to a gradient of soluble CXCL12 in transwell assays. CONCLUSIONS: Collectively, these studies demonstrate that HCMV impairs EVT migration and invasion induced by CXCL12. As HCMV has the ability to inhibit EVT migration and invasion through dysregulation of other relevant signaling pathways, it is likely that the virus affects multiple signaling pathways to impair placentation and contribute to some of the placental defects seen in HCMV-positive pregnancies.
Asunto(s)
Quimiocina CXCL12/metabolismo , Infecciones por Citomegalovirus/metabolismo , Citomegalovirus , Trofoblastos/metabolismo , Trofoblastos/virología , Línea Celular , Movimiento Celular/genética , Quimiocina CXCL12/genética , Infecciones por Citomegalovirus/genética , Femenino , Expresión Génica , Humanos , Embarazo , ARN Mensajero/genética , Receptores CXCR/genética , Receptores CXCR/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismoRESUMEN
BACKGROUND: Endothelial barrier dysfunction (EBD) involves microtubule disassembly and enhanced cell contractility. Histone deacetylase 6 (HDAC6) deacetylates α-tubulin, and thereby destabilizes microtubules. This study investigates a role for HDAC6 in EBD. METHODS: EBD was induced with thrombin±HDAC6 inhibitors (tubacin and MC1575), and assessed by transendothelial electrical resistance (TEER). Markers for microtubule disassembly (α-tubulin and acetylated α-tubulin) and contraction (phosphorylated myosin light chain 2, P-MLC2) were measured using immunoblots and immunofluorescence. RESULTS AND CONCLUSION: Thrombin induced a â¼50% decrease in TEER that was abrogated by the HDAC6 inhibitors. Moreover, inhibition of HDAC6 diminished edema in the lung injured by lipopolysaccharide. Lastly, inhibition of HDAC6 attenuated thrombin-induced microtubule disassembly and P-MLC2. Our results suggest that HDAC6 can be targeted to limit EBD.
Asunto(s)
Endotelio Vascular/enzimología , Histona Desacetilasas/metabolismo , Microtúbulos/metabolismo , Edema Pulmonar/enzimología , Acetilación , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Permeabilidad , Trombina/farmacologíaRESUMEN
Composition of extracellular matrix (ECM) is crucial to the establishment and maintenance of epithelial apical-basolateral polarity. Increased ECM rigidity caused by deposition of fibrillar collagen, for example, collagen type I (Col-1), promotes loss of epithelial polarity and tumor progression. microRNAs are small non-coding RNAs that regulate gene expression and fundamental cellular processes. The current study explored a link between microRNAs and Col-1 using organotypic three-dimensional culture in which epithelial cells are embedded within Matrigel, a mimic of basement membrane matrix (Matrigel 3-D). Matrigel 3-D culture of A549, MCF-7, and mK-ras-LE cells (lung and mammary epithelial cell lines) gave rise to acinus, an in vitro equivalent of apical-basolateral polarity that consists of a polarized monolayer of epithelial cells facing a central lumen. Supplementation of Col-1 disrupted acinus. Moreover, Col-1 up-regulated the expression of miR-21, a well-documented oncogenic microRNA, via a post-transcriptional mechanism. Similar post-transcriptional up-regulation of miR-21 correlated with deposition of Col-1 in a murine model of lung fibrogenesis. In summary, our findings link altered ECM composition/rigidity and the expression of oncogenic microRNAs. The current study also suggests a novel post-transcriptional mechanism for regulation of miR-21 expression at maturation from pre-miR-21 to mature miR-21.
Asunto(s)
Colágeno Tipo I/fisiología , MicroARNs/fisiología , Procesamiento Postranscripcional del ARN , Regulación hacia Arriba , Animales , Línea Celular Tumoral , Humanos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Human cytomegalovirus (HCMV) is a near ubiquitous herpesvirus that relies on host cell metabolism for efficient replication. Although it has been shown that HCMV requires functional host cell mitochondria for efficient replication, it is unknown whether mitochondrial targeted pharmacological agents can be repurposed as antivirals. Here we report that treatment with drugs targeting the electron transport chain (ETC) complexes inhibit HCMV replication. Addition of rotenone, oligomycin, antimycin and metformin resulted in decreased HCMV titers in vitro, independent of HCMV strain. This further illustrates the dependence of HCMV replication on functional mitochondria. Metformin, an FDA approved drug, delays HCMV replication kinetics resulting in a reduction of viral titers. Repurposing metformin as an antiviral is advantageous as its safety profile and epidemiological data are well accepted. Our findings provide new insight into the potential for targeting HCMV infection through host cell metabolism and how these pharmacological interventions function.
Asunto(s)
Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Transporte de Electrón/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Prepucio/citología , Humanos , Masculino , Metformina/farmacología , Oligomicinas/farmacologíaRESUMEN
Several studies have implicated gamma-herpesviruses, particularly Epstein-Barr virus (EBV), in the progression of idiopathic pulmonary fibrosis. The data presented here examine the possible role that EBV plays in the potentiation of this disease by evaluating the pulmonary response to expression of the EBV lytic transactivator protein Zta. Expression of Zta in the lungs of mice via adenovirus-mediated delivery (Adv-Zta) produced profibrogenic inflammation that appeared most pronounced by day 7 postexposure. Relative to mice exposed to control GFP-expressing adenovirus (Adv-GFP), mice exposed to Adv-Zta displayed evidence of lung injury and a large increase in inflammatory cells, predominantly neutrophils, recovered by bronchoalveolar lavage (BAL). Cytokine and mRNA profiling of the BAL fluid and cells recovered from Adv-Zta-treated mice revealed a Th2 and Th17 bias. mRNA profiles from Adv-Zta-infected lung epithelial cells revealed consistent induction of mRNAs encoding Th2 cytokines. Coexpression in transient assays of wild-type Zta, but not a DNA-binding-defective mutant Zta, activated expression of the IL-13 promoter in lung epithelial cells, and detection of IL-13 in Adv-Zta-treated mice correlated with expression of Zta. Induction of Th2 cytokines in Zta-expressing mice corresponded with alternative activation of macrophages. In cell culture and in mice, Zta repressed lung epithelial cell markers. Despite the profibrogenic character at day 7, the inflammation resolves by 28 days postexposure to Adv-Zta without evidence of fibrosis. These observations indicate that the EBV lytic transactivator protein Zta displays activity consistent with a pathogenic role in pulmonary fibrosis associated with herpesvirus infection.
Asunto(s)
Herpesvirus Humano 4/metabolismo , Neumonía , Transactivadores/metabolismo , Animales , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Línea Celular , Citocinas/inmunología , Humanos , Activación de Macrófagos , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Neumonía/inmunología , Neumonía/patología , Neumonía/virología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genéticaRESUMEN
Multipotent stromal cells (MSCs) have been shown to reduce apoptosis in injured cells by secretion of paracrine factors, but these factors were not fully defined. We observed that coculture of MSCs with previously UV-irradiated fibroblasts reduced apoptosis of the irradiated cells, but fresh MSC conditioned medium was unable reproduce the effect. Comparative microarray analysis of MSCs grown in the presence or absence of UV-irradiated fibroblasts demonstrated that the MSCs were activated by the apoptotic cells to increase synthesis and secretion of stanniocalcin-1 (STC-1), a peptide hormone that modulates mineral metabolism and has pleiotrophic effects that have not been fully characterized. We showed that STC-1 was required but not sufficient for reduction of apoptosis of UV-irradiated fibroblasts. In contrast, we demonstrated that MSC-derived STC-1 was both required and sufficient for reduction of apoptosis of lung cancer epithelial cells made apoptotic by incubation at low pH in hypoxia. Our data demonstrate that STC-1 mediates the antiapoptotic effects of MSCs in two distinct models of apoptosis in vitro.
Asunto(s)
Apoptosis , Glicoproteínas/fisiología , Células del Estroma/citología , Células del Estroma/metabolismo , Regulación hacia Arriba , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , Supervivencia Celular , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Glicoproteínas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacos , Transfección , Rayos UltravioletaRESUMEN
Aging is associated with a decline in immune function that is not fully understood including vaccine failure. Here we report transcriptomic analysis on B cells from naive or influenza-vaccinated mice of 3 ages: young (15-23 weeks), middle-aged (63-81 weeks), and old mice (103-119 weeks). Our goal was expression profiling of B cells by age and history of vaccination to identify novel changes at the transcriptome level. We observed waning vaccine responses with age. In B cell transcripts, age and vaccination history were both important with notable differences observed in conducted analyses (eg, principal component, gene set enrichment, differentially expressed [DE] genes, and canonical pathways). Only 39 genes were significantly DE with age irrespective of vaccine history. This included age-related changes to box C/D small nucleolar (sno) RNAs, Snord123 and Snord1a. Box C/D snoRNAs regulate rRNAs through methylation and are linked to neurodegenerative, inflammatory, and cancer diseases but not specifically B cells or age. Canonical pathway changes implicated with age irrespective of vaccination history included EIF2, mTOR signaling, p53, Paxillin, and Tec kinase signaling pathways as well as cell cycle checkpoint. Importantly, we identified DE genes and pathways that were progressively altered starting in middle-age (eg, signaling by Rho family GTPases) or only altered in middle-age (eg, sphingosine-1-phosphate signaling), despite minimal differences in the ability of these mice to respond to vaccination compared to younger mice. Our results indicate the importance of vaccination or immune stimulation and analyses of multiple age ranges for aging B cell studies and validate an experimental model for future studies.
Asunto(s)
Linfocitos B/inmunología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Perfilación de la Expresión Génica , Vacunas contra la Influenza/inmunología , ARN Nucleolar Pequeño/genética , Factores de Edad , Animales , Ratones , Bazo/citologíaRESUMEN
Tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta(1) (TGF-beta(1)) are peptides with multiple biological activities that influence neoplastic, immunologic and fibroproliferative diseases. There are clear interrelationships and overlap between the actions of TNF-alpha and TGF-beta(1) in lung fibrosis; therefore, we postulated that TNF-alpha may play a significant role in regulating TGF-beta(1) expression in lungs. We recently reported that TNF-alpha activates the extracellular regulated kinase (ERK)-specific pathway in fibroblasts resulting in stabilization of TGF-beta(1) mRNA and increased expression of TGF-beta(1). In the current study, we further investigated the molecular mechanisms involved in TNF-alpha regulation of TGF-beta(1) expression. Nuclear run-on assays showed that treatment of Swiss 3T3 fibroblasts with TNF-alpha increased transcription of the TGF-beta(1) gene in an ERK independent manner. Pre-treatment with the activator protein-1 (AP-1) inhibitor curcumin attenuated TNF-alpha induced transcription of the TGF-beta(1) gene. TNF-alpha induced increased levels of c-Jun and C-Fos in the nucleus accompanied by phosphorylation of c-Jun. In electrophoretic mobility shift assays, AP-1 binding to an AP-1 binding site found within the TGF-beta(1) promoter was increased in nuclear extracts from Swiss 3T3 fibroblasts treated with TNF-alpha. Together, these results suggest that TNF-alpha induces expression and DNA binding of AP-1 resulting in increased transcription of the TGF-beta(1) gene. It is essential to know which transcription pathways are activated because of the wide distribution of TNF-alpha and TGF-beta(1), the general lack of effective treatments for fibroproliferative disease and the possibility that targeting the correct transcription factors could be palliative.
Asunto(s)
Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Células 3T3 , Animales , Secuencia de Bases , Cartilla de ADN , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Infusion of bone marrow stem or progenitor cells may provide powerful therapies for injured tissues such as the lung and heart. We examined the potential of bone marrow-derived (BMD) progenitor cells to contribute to repair and remodeling of lung and heart in a rat monocrotaline (MCT) model of pulmonary hypertension. Bone marrow from green fluorescent protein (GFP)-transgenic male rats was transplanted into GFP-negative female rats. The chimeric animals were injected with MCT to produce pulmonary hypertension. Significant numbers of male GFP-positive BMD cells engrafted in the lungs of MCT-treated rats. Microarray analyses and double-immunohistochemistry demonstrated that many of the cells were interstitial fibroblasts or myofibroblasts, some of the cells were hematopoietic cells, and some were pulmonary epithelial cells (Clara cells), vascular endothelial cells, and smooth muscle cells. A few BMD cells fused with pulmonary cells from the host, but the frequency was low. In the hypertrophied hearts of MCT-treated rats, we found a significant increase in the relative numbers of BMD cells in the right ventricle wall as compared with the left ventricle. Some of the BMD cells in the right ventricle were vascular cells and cardiomyocytes. We report BMD cardiomyocytes with a normal chromosome number, fusion of BMD cells with host cardiomyocytes, and, in some cases, nuclear fusion.
Asunto(s)
Células de la Médula Ósea/citología , Hipertensión Pulmonar/terapia , Pulmón/citología , Miocardio/citología , Células Madre/citología , Cicatrización de Heridas , Animales , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Diferenciación Celular , Modelos Animales de Enfermedad , Femenino , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/metabolismo , Corazón/fisiología , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/patología , Pulmón/metabolismo , Masculino , Monocrotalina , Ratas , Células Madre/metabolismoRESUMEN
BACKGROUND: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS: In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3beta as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3beta. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3beta phosphorylation and the subsequent expression of beta-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of beta-catenin alone was insufficient to stimulate cell motility.