RESUMEN
Herpes simplex virus type 1 (HSV-1), a neurotropic DNA virus, establishes latency in neural tissues, with reactivation causing severe consequences like encephalitis. Emerging evidence links HSV-1 infection to chronic neuroinflammation and neurodegenerative diseases. Microglia, the central nervous system's (CNS) immune sentinels, express diverse receptors, including α7 nicotinic acetylcholine receptors (α7 nAChRs), critical for immune regulation. Recent studies suggest α7 nAChR activation protects against viral infections. Here, we show that α7 nAChR agonists, choline and PNU-282987, significantly inhibit HSV-1 replication in microglial BV2 cells. Notably, this inhibition is independent of the traditional ionotropic nAChR signaling pathway. mRNA profiling revealed that choline stimulates the expression of antiviral factors, IL-1ß and Nos2, and down-regulates the apoptosis genes and type A Lamins in BV2 cells. These findings suggest a novel mechanism by which microglial α7 nAChRs restrict viral infections by regulating innate immune responses.
Asunto(s)
Colina , Herpesvirus Humano 1 , Microglía , Replicación Viral , Receptor Nicotínico de Acetilcolina alfa 7 , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genética , Microglía/virología , Microglía/efectos de los fármacos , Microglía/metabolismo , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/efectos de los fármacos , Animales , Línea Celular , Ratones , Replicación Viral/efectos de los fármacos , Colina/farmacología , Colina/metabolismo , Compuestos Bicíclicos con Puentes/farmacología , Benzamidas/farmacología , Inmunidad Innata , Herpes Simple/virología , Herpes Simple/metabolismo , Interleucina-1beta/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Antivirales/farmacología , Agonistas Nicotínicos/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo II/genéticaRESUMEN
Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25-2.5 µM cisplatin (CisPt) or 12.5-100 µM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 µM CisPt from 3 to 96 h expressing 2000-5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity.
Asunto(s)
Células Epiteliales , Túbulos Renales Proximales , Humanos , Ratones , Animales , RNA-Seq , Línea Celular , Túbulos Renales Proximales/metabolismo , Cisplatino/metabolismoRESUMEN
Individuals infected by SARS-CoV-2 are at risk of developing neurological-related post-acute disorders. Disputed epidemiological data indicated nicotine may reduce the severity of infection. Here we find exposure to nicotine in drinking water does not alter the moribundity of hACE2 mice. However, pre-exposure to nicotine decreased the likelihood of SARS-CoV-2 RNA expression and pathology in the brain. These results suggest mechanisms involving targets of nicotine could be leveraged to prevent the neurovirulence of SARS-CoV-2.
Asunto(s)
COVID-19 , Enfermedades del Sistema Nervioso , Ratones , Animales , SARS-CoV-2 , COVID-19/patología , Pulmón/patología , ARN Viral , Nicotina/farmacología , Ratones Transgénicos , Enfermedades del Sistema Nervioso/patología , Encéfalo , Modelos Animales de EnfermedadRESUMEN
Individuals infected by SARS-CoV-2 are at risk of developing neurological-related post-acute disorders. Disputed epidemiological data indicated nicotine may reduce the severity of infection. Here we find exposure to nicotine in drinking water does not alter the moribundity of hACE2 mice. However, pre-exposure to nicotine decreased the likelihood of SARS-CoV-2 RNA expression and pathology in the brain. These results suggest mechanisms involving targets of nicotine could be leveraged to prevent the neurovirulence of SARS-CoV-2.
RESUMEN
Cadmium (Cd) is one of the most prevalent environmental heavy metal contaminants and is considered an endocrine disruptor and carcinogen. In women with uterine fibroids, there is a correlation between blood Cd levels and fibroid tumor size. In this study, fibroid cells were exposed to 10 µM CdCl2 for 6 months and a fast-growing Cd-Resistant Leiomyoma culture, termed CR-LM6, was recovered. To characterize the morphological and mechanodynamic features of uterine fibroid cells associated with prolonged Cd exposure, we conducted time lapse imaging using a Zeiss confocal microscope and analyzed data by Imaris and RStudio. Our experiments recorded more than 64,000 trackable nuclear surface objects, with each having multiple parameters such as nuclear size and shape, speed, location, orientation, track length, and track straightness. Quantitative analysis revealed that prolonged Cd exposure significantly altered cell migration behavior, such as increased track length and reduced track straightness. Cd exposure also significantly increased the heterogeneity in nuclear size. Additionally, Cd significantly increased the median and variance of instantaneous speed, indicating that Cd exposure results in higher speed and greater variation in motility. Profiling of mRNA by NanoString analysis and Ingenuity Pathway Analysis (IPA) strongly suggested that the direction of gene expression changes due to Cd exposure enhanced cell movement and invasion. The altered expression of extracellular matrix (ECM) genes such as collagens, matrix metallopeptidases (MMPs), secreted phosphoprotein 1 (SPP1), which are important for migration contact guidance, may be responsible for the greater heterogeneity. The significantly increased heterogeneity of nuclear size, speed, and altered migration patterns may be a prerequisite for fibroid cells to attain characteristics favorable for cancer progression, invasion, and metastasis.
RESUMEN
The heavy metal Cadmium (Cd), a widespread environmental contaminant, poses serious hazards to human health and is considered a metallohormone and carcinogen. In women with uterine fibroids, there is a significant association between blood Cd levels and increased fibroid tumor size. The aim of this study was to determine if benign human uterine leiomyoma (fibroid) cells could be malignantly transformed in vitro by continuous Cd exposure and, if so, explore a molecular mechanism by which this could occur. We found when fibroid cells were exposed to 10 µM CdCl2 for 8 weeks, a robust and fast-growing Cd-Resistant Leiomyoma (CR-LM) cell culture was established. The CR-LM cells formed viable colonies in soft agar and had increased cytoplasmic glycogen aggregates, enhanced cell motility, a higher percentage of cells in G2/M phase, and increased expression of the proliferation marker Ki-67. NanoString analysis showed downregulation of genes encoding for extracellular matrix (ECM) components, such as collagens, fibronectins, laminins, and SLRP family proteins, whereas genes involved in ECM degradation (MMP1, MMP3, and MMP10) were significantly upregulated. A volcano plot showed that the top differentially genes favored cancer progression. Functional analysis by ingenuity pathway analysis predicted a significant inhibition of TGFB1 signaling, leading to enhanced proliferation and attenuated fibrosis. Prolonged Cd exposure altered phenotypic characteristics and dysregulated genes in fibroid cells predicative of progression towards a cancer phenotype. Therefore, continuous Cd exposure alters the benign characteristics of fibroid cells in vitro, and Cd exposure could possibly pose a health hazard for women with uterine fibroids.
Asunto(s)
Cadmio/toxicidad , Matriz Extracelular/metabolismo , Leiomioma/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias Uterinas/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Leiomioma/patología , Neoplasias Uterinas/patologíaRESUMEN
Viral agents are of interest as possible autoimmune triggers due to prior reported associations and widely studied molecular mechanisms of antiviral immune responses in autoimmunity. Here we examined new viral candidates for the initiation and/or promotion of systemic autoimmune diseases (SAID), as well as possible related signaling pathways shared in the pathogenesis of those disorders. RNA isolated from peripheral blood samples from 33 twins discordant for SAID and 33 matched, unrelated healthy controls was analyzed using a custom viral-human gene microarray. Paired comparisons were made among three study groups-probands with SAID, their unaffected twins, and matched, unrelated healthy controls-using statistical and molecular pathway analyses. Probands and unaffected twins differed significantly in the expression of 537 human genes, and 107 of those were associated with viral infections. These 537 differentially expressed human genes participate in overlapping networks of several canonical, biologic pathways relating to antiviral responses and inflammation. Moreover, certain viral genes were expressed at higher levels in probands compared to either unaffected twins or unrelated, healthy controls. Interestingly, viral gene expression levels in unaffected twins appeared intermediate between those of probands and the matched, unrelated healthy controls. Of the viruses with overexpressed viral genes, herpes simplex virus-2 (HSV-2) was the only human viral pathogen identified using four distinct oligonucleotide probes corresponding to three HSV-2 genes associated with different stages of viral infection. Although the effects from immunosuppressive therapy on viral gene expression remain unclear, this exploratory study suggests a new approach to evaluate shared viral agents and antiviral immune responses that may be involved in the development of SAID.
Asunto(s)
Enfermedades Autoinmunes/genética , Genes Virales , Herpesvirus Humano 2 , Gemelos/genética , Adolescente , Enfermedades Autoinmunes/virología , Autoinmunidad/genética , Niño , Preescolar , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Transcriptoma , Adulto JovenRESUMEN
INTRODUCTION: The objective of this study is to determine if multiple systemic autoimmune diseases (SAID) share gene expression pathways that could provide insights into pathogenic mechanisms common to these disorders. METHODS: RNA microarray analyses (Agilent Human 1A(V2) 20K oligo arrays) were used to quantify gene expression in peripheral blood cells from 20 monozygotic (MZ) twin pairs discordant for SAID. Six affected probands with systemic lupus erythematosus (SLE), six with rheumatoid arthritis (RA), eight with idiopathic inflammatory myopathies (IIM), and their same-gendered unaffected twins, were enrolled. Comparisons were made between discordant twin pairs and these were also each compared to 40 unrelated control subjects (matched 2:1 to each twin by age, gender and ethnicity) using statistical and molecular pathway analyses. Relative quantitative PCR was used to verify independently measures of differential gene expression assessed by microarray analysis. RESULTS: Probands and unrelated, matched controls differed significantly in gene expression for 104 probes corresponding to 92 identifiable genes (multiple-comparison adjusted P values < 0.1). Differentially expressed genes involved several overlapping pathways including immune responses (16%), signaling pathways (24%), transcription/translation regulators (26%), and metabolic functions (15%). Interferon (IFN)-response genes (IFI27, OASF, PLSCR1, EIF2AK2, TNFAIP6, and TNFSF10) were up-regulated in probands compared to unrelated controls. Many of the abnormally expressed genes played regulatory roles in multiple cellular pathways. We did not detect any probes expressed differentially in comparisons among the three SAID phenotypes. Similarly, we found no significant differences in gene expression when comparing probands to unaffected twins or unaffected twins to unrelated controls. Gene expression levels for unaffected twins appeared intermediate between that of probands and unrelated controls for 6535 probes (32% of the total probes) as would be expected by chance. By contrast, in unaffected twins intermediate ordering was observed for 84 of the 104 probes (81%) whose expression differed significantly between probands and unrelated controls. CONCLUSIONS: Alterations in expression of a limited number of genes may influence the dysregulation of numerous, integrated immune response, cell signaling and regulatory pathways that are common to a number of SAID. Gene expression profiles in peripheral blood suggest that for genes in these critical pathways, unaffected twins may be in a transitional or intermediate state of immune dysregulation between twins with SAID and unrelated controls, perhaps predisposing them to the development of SAID given the necessary and sufficient environmental exposures.
Asunto(s)
Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades en Gemelos/genética , Perfilación de la Expresión Génica , Análisis por Conglomerados , Enfermedades en Gemelos/inmunología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Reacción en Cadena en Tiempo Real de la Polimerasa , Gemelos Monocigóticos/genéticaRESUMEN
The neurodegenerative disease Friedreich's ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials.
Asunto(s)
Daño del ADN , Ataxia de Friedreich/genética , Ataxia de Friedreich/patología , Expresión Génica , ARN/sangre , Adolescente , Adulto , Células Cultivadas , Niño , Estudios de Cohortes , Femenino , Humanos , Proteínas de Unión a Hierro/genética , Masculino , Persona de Mediana Edad , ARN/genética , Adulto Joven , FrataxinaRESUMEN
UNLABELLED: The diagnosis and management of drug-induced liver injury (DILI) is hindered by the limited utility of traditional clinical chemistries. It has recently been shown that hepatotoxicants can produce compound-specific changes in the peripheral blood (PB) transcriptome in rodents, suggesting that the blood transcriptome might provide new biomarkers of DILI. To investigate in humans, we used DNA microarrays as well as serum metabolomic methods to characterize changes in the transcriptome and metabolome in serial PB samples obtained from six healthy adults treated with a 4-g bolus dose of acetaminophen (APAP) and from three receiving placebo. Treatment did not cause liver injury as assessed by traditional liver chemistries. However, 48 hours after exposure, treated subjects showed marked down-regulation of genes involved in oxidative phosphorylation/mitochondrial function that was not observed in the placebos (P < 1.66E-19). The magnitude of down-regulation was positively correlated with the percent of APAP converted to the reactive metabolite N-acetyl-p-benzoquinone-imide (NAPQI) (r= 0.739;P= 0.058). In addition, unbiased analysis of the serum metabolome revealed an increase in serum lactate from 24 to 72 hours postdosing in the treated subjects alone (P< 0.005). Similar PB transcriptome changes were observed in human overdose patients and rats receiving toxic doses. CONCLUSION: The single 4-g APAP dose produced a transcriptome signature in PB cells characterized by down-regulation of oxidative phosphorylation genes accompanied by increased serum lactate. Similar gene expression changes were observed in rats and several patients after consuming hepatotoxic doses of APAP. The timing of the changes and the correlation with NAPQI production are consistent with mechanisms known to underlie APAP hepatoxicity. These studies support the further exploration of the blood transcriptome for biomarkers of DILI.
Asunto(s)
Acetaminofén/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Metaboloma/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Acetaminofén/orina , Adulto , Animales , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Hígado/metabolismo , Persona de Mediana Edad , Placebos , RatasRESUMEN
Microarrays have been used to evaluate the expression of thousands of genes in various tissues. However, few studies have investigated the change in gene expression profiles in one of the most easily accessible tissues, whole blood. We utilized an acute inflammation model to investigate the possibility of using a cDNA microarray to measure the gene expression profile in the cells of whole blood. Blood was collected from male Sprague-Dawley rats at 2 and 6 h after treatment with 5 mg/kg (ip) LPS. Hematology showed marked neutrophilia accompanied by lymphopenia at both time points. TNF-alpha and IL-6 levels were markedly elevated at 2 h, indicating acute inflammation, but by 6 h the levels had declined. Total RNA was isolated from whole blood and hybridized to the National Institute of Environmental Health Sciences Rat Chip v.3.0. LPS treatment caused 226 and 180 genes to be differentially expressed at 2 and 6 h, respectively. Many of the differentially expressed genes are involved in inflammation and the acute phase response, but differential expression was also noted in genes involved in the cytoskeleton, cell adhesion, oxidative respiration, and transcription. Real-time RT-PCR confirmed the differential regulation of a representative subset of genes. Principal component analysis of gene expression discriminated between the acute inflammatory response apparent at 2 h and the observed recovery underway at 6 h. These studies indicate that, in whole blood, changes in gene expression profiles can be detected that are reflective of inflammation, despite the adaptive shifts in leukocyte populations that accompany such inflammatory processes.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Inflamación/sangre , Lipopolisacáridos/metabolismo , Animales , Adhesión Celular , Análisis por Conglomerados , ADN Complementario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Masculino , Muridae , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxígeno/metabolismo , Análisis de Componente Principal , ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
OBJECTIVES: Cyclooxygenase-1 and 2 (COX-1 and COX-2) play important roles in normal physiology and are often dysregulated in neoplastic tissues. The present study determines whether COX-1 and COX-2 are expressed in ovarian cancers and whether the pattern of expression of these enzymes reveals clues to their roles in this cancer. METHODS: The expression of COX-1 and COX-2 proteins in 9 normal human ovaries, in 137 cases of ovarian cancers of epithelial origin (83 primary and 54 metastatic), and in 7 ovarian cancer cell lines was examined by immunohistochemistry and western analysis. RESULTS: COX-1 protein was present in 95/137 (69.3%) of the total cancers studied, with 55/83 (66.3%) of the primary cancers and 40/54 (74.1%) of the metastatic cancers positive for protein. COX-2 was present in 97/137 (70.8%) of all cancers studied, with 53/83 (63.9%) of the primary cancers and 44/54 (81.5%) of the metastatic cancers positive for protein. Notably, the quickscores for COX-2-positive staining were significantly higher in metastatic cancers. Moreover, COX-2 immunostaining was frequently found at the advancing margin of tumor invasion or in new metastatic loci. COX-1 protein expression was observed in the ovarian surface epithelial cells, especially that of the inclusion cysts. COX-1 was also detected by western blot in seven of nine ovarian cancer cell lines. However, no COX-2 was detected in either normal epithelium or cancer cell lines. CONCLUSION: COX-1 and COX-2 were expressed in every type of ovarian epithelial cancer, suggesting that each may contribute to the cancer development or progression.