RESUMEN
MicroRNAs (miR) are a group of small, non-coding RNAs of 17-25 nucleotides that regulate gene expression at the post-transcriptional level. Dysregulation of miRNA expression or function may contribute to abnormal gene expression and signaling pathways, leading to disease pathology. Lagovirus europaeus (L. europaeus) causes severe disease in rabbits called rabbit hemorrhagic disease (RHD). The symptoms of liver, lung, kidney, and spleen degeneration observed during RHD are similar to those of acute liver failure (ALF) and multi-organ failure (MOF) in humans. In this study, we assessed the expression of miRs and their target genes involved in the innate immune and inflammatory response. Also, we assessed their potential impact on pathways in L. europaeus infection-two genotypes (GI.1 and GI.2)-in the liver, lungs, kidneys, and spleen. The expression of miRs and target genes was determined using quantitative real-time PCR (qPCR). We assessed the expression of miR-155 (MyD88, TAB2, p65, NLRP3), miR-146a (IRAK1, TRAF6), miR-223 (TLR4, IKKα, NLRP3), and miR-125b (MyD88). We also examined biomarkers of inflammation: IL-1ß, IL-6, TNF-α, and IL-18 in four tissues at the mRNA level. Our study shows that the main regulators of the innate immune and inflammatory response in L. europaeus/GI.1 and GI.2 infection, as well as RHD, are miR-155, miR-223, and miR-146a. During infection with L. europaeus/RHD, miR-155 has both pro- and anti-inflammatory effects in the liver and anti-inflammatory effects in the kidneys and spleen; miR-146a has anti-inflammatory effects in the liver, lungs and kidneys; miR-223 has anti-inflammatory effects in all tissues; however, miR-125b has anti-inflammatory effects only in the liver. In each case, such an effect may be a determinant of the pathogenesis of RHD. Our research shows that miRs may regulate three innate immune and inflammatory response pathways in L. europaeus infection. However, the result of this regulation may be influenced by the tissue microenvironment. Our research shows that infection of rabbits with L. europaeus/GI.1 and GI.2 genotypes causes an overexpression of two critical acute phase cytokines: IL-6 in all examined tissues and TNF-α (in the liver, lungs, and spleen). IL-1ß was highly expressed only in the lungs after L. europaeus infection. These facts indicate a strong and rapid involvement of the local innate immune and inflammatory response in L. europaeus infection-two genotypes (GI.1 and GI.2)-and in the pathogenesis of RHD. Profile of biomarkers of inflammation in rabbits infected with L. europaeus/GI.1 and GI.2 genotypes are similar regarding the nature of changes but are different for individual tissues. Therefore, we propose three inflammation profiles for L. europaeus infection for both GI.1 and GI.2 genotypes (pulmonary, renal, liver, and spleen).
Asunto(s)
Infecciones por Caliciviridae , Genotipo , Virus de la Enfermedad Hemorrágica del Conejo , Inmunidad Innata , MicroARNs , Animales , MicroARNs/genética , Inmunidad Innata/genética , Conejos , Infecciones por Caliciviridae/genética , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/virología , Virus de la Enfermedad Hemorrágica del Conejo/genética , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Inflamación/genética , Inflamación/inmunología , Regulación de la Expresión Génica , Hígado/metabolismo , Hígado/patología , Hígado/virologíaRESUMEN
Baculovirus is an obligate parasitic virus of the phylum Arthropoda. Baculovirus including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used in the laboratory and industrial preparation of proteins or protein complexes. Due to its large packaging capacity and non-replicative and non-integrative natures in mammals, baculovirus has been proposed as a gene therapy vector for transgene delivery. However, the mechanism of baculovirus transduction in mammalian cells has not been fully illustrated. Here, we employed a cell surface protein-focused CRISPR screen to identify host dependency factors for baculovirus transduction in mammalian cells. The screening experiment uncovered a series of baculovirus host factors in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and NPC intracellular cholesterol transporter 1 (NPC1). Further investigation illustrated that EXTL3 affected baculovirus attachment and entry by participating in heparan sulfate biosynthesis. In addition, NPC1 promoted baculovirus transduction by mediating membrane fusion and endosomal escape. Moreover, in vivo, baculovirus transduction in Npc1-/+ mice showed that disruption of Npc1 gene significantly reduced baculovirus transduction in mouse liver. In summary, our study revealed the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape in mammalian cells, which is useful for understanding baculovirus transduction in human cells.
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N-Acetilglucosaminiltransferasas , Proteína Niemann-Pick C1 , Nucleopoliedrovirus , Animales , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/fisiología , Humanos , Ratones , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/genética , Células HEK293 , Endosomas/metabolismo , Heparitina Sulfato/metabolismo , Internalización del Virus , Transducción Genética , Células Sf9 , Hígado/metabolismo , Hígado/virología , Sistemas CRISPR-CasRESUMEN
Chronic liver disease and cancer are global health challenges. The role of the circadian clock as a regulator of liver physiology and disease is well established in rodents, however, the identity and epigenetic regulation of rhythmically expressed genes in human disease is less well studied. Here we unravel the rhythmic transcriptome and epigenome of human hepatocytes using male human liver chimeric mice. We identify a large number of rhythmically expressed protein coding genes in human hepatocytes of male chimeric mice, which includes key transcription factors, chromatin modifiers, and critical enzymes. We show that hepatitis C virus (HCV) infection, a major cause of liver disease and cancer, perturbs the transcriptome by altering the rhythmicity of the expression of more than 1000 genes, and affects the epigenome, leading to an activation of critical pathways mediating metabolic alterations, fibrosis, and cancer. HCV-perturbed rhythmic pathways remain dysregulated in patients with advanced liver disease. Collectively, these data support a role for virus-induced perturbation of the hepatic rhythmic transcriptome and pathways in cancer development and may provide opportunities for cancer prevention and biomarkers to predict HCC risk.
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Ritmo Circadiano , Hepacivirus , Hepatitis C , Hepatocitos , Hígado , Transcriptoma , Humanos , Hígado/metabolismo , Hígado/virología , Animales , Masculino , Hepatocitos/metabolismo , Hepatocitos/virología , Ratones , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C/genética , Hepatitis C/metabolismo , Hepatitis C/virología , Ritmo Circadiano/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Neoplasias Hepáticas/metabolismo , Relojes Circadianos/genética , Epigénesis GenéticaRESUMEN
BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD), characterised by hepatic lipid accumulation, causes inflammation and oxidative stress accompanied by cell damage and fibrosis. Liver injury (LI) is also frequently reported in patients hospitalised with coronavirus disease 2019 (COVID-19), while pre-existing MASLD increases the risk of LI and the development of COVID-19-associated cholangiopathy. Mechanisms of injury at the cellular level remain unclear, but it may be significant that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes COVID-19, uses angiotensin-converting expression enzyme 2 (ACE2), a key regulator of the 'anti-inflammatory' arm of the renin-angiotensin system, for viral attachment and host cell invasion. AIM: To determine if hepatic ACE2 levels are altered during progression of MASLD and in patients who died with severe COVID-19. METHODS: ACE2 protein levels and localisation, and histological fibrosis and lipid droplet accumulation as markers of MASLD were determined in formalin-fixed liver tissue sections across the MASLD pathological spectrum (isolated hepatocellular steatosis, metabolic dysfunction-associated steatohepatitis (MASH) +/- fibrosis, end-stage cirrhosis) and in post-mortem tissues from patients who had died with severe COVID-19, using ACE2 immunohistochemistry and haematoxylin and eosin and picrosirius red staining of total collagen and lipid droplet areas, followed by quantification using machine learning-based image pixel classifiers. RESULTS: ACE2 staining is primarily intracellular and concentrated in the cytoplasm of centrilobular hepatocytes and apical membranes of bile duct cholangiocytes. Strikingly, ACE2 protein levels are elevated in non-fibrotic MASH compared to healthy controls but not in the progression to MASH with fibrosis and in cirrhosis. ACE2 protein levels and histological fibrosis are not associated, but ACE2 and liver lipid droplet content are significantly correlated across the MASLD spectrum. Hepatic ACE2 levels are also increased in COVID-19 patients, especially those showing evidence of LI, but are not correlated with the presence of SARS-CoV-2 virus in the liver. However, there is a clear association between the hepatic lipid droplet content and the presence of the virus, suggesting a possible functional link. CONCLUSION: Hepatic ACE2 levels were elevated in nonfibrotic MASH and COVID-19 patients with LI, while lipid accumulation may promote intra-hepatic SARS-CoV-2 replication, accelerating MASLD progression and COVID-19-mediated liver damage.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Hígado Graso , Hígado , SARS-CoV-2 , Humanos , COVID-19/complicaciones , COVID-19/mortalidad , COVID-19/patología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/análisis , Masculino , Hígado/patología , Hígado/enzimología , Hígado/virología , Femenino , SARS-CoV-2/patogenicidad , Persona de Mediana Edad , Hígado Graso/patología , Hígado Graso/virología , Hígado Graso/enzimología , Hígado Graso/mortalidad , Anciano , Adulto , Cirrosis Hepática/patología , Cirrosis Hepática/virología , Cirrosis Hepática/mortalidad , Cirrosis Hepática/enzimología , Progresión de la EnfermedadRESUMEN
Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are manifestations of the post-acute sequelae of SARS-CoV-2. Our understanding of metabolic decline after acute COVID-19 remains unclear due to the lack of animal models. Here, we report a non-human primate model of metabolic post-acute sequelae of SARS-CoV-2 using SARS-CoV-2 infected African green monkeys. Using this model, we identify a dysregulated blood chemokine signature during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. Hyperglycemia also correlates with liver glycogen levels, but there is no evidence of substantial long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the African green monkey model exhibits important similarities to humans and can be utilized to assess therapeutic candidates to combat COVID-related metabolic defects.
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COVID-19 , Modelos Animales de Enfermedad , Hiperglucemia , Hígado , SARS-CoV-2 , Animales , Hiperglucemia/inmunología , COVID-19/inmunología , COVID-19/virología , COVID-19/sangre , Chlorocebus aethiops , SARS-CoV-2/inmunología , Hígado/virología , Hígado/metabolismo , Hígado/inmunología , Glucógeno/metabolismo , Glucemia/metabolismo , Humanos , Masculino , Páncreas/virología , Páncreas/inmunología , Páncreas/patología , Páncreas/metabolismo , Quimiocinas/metabolismo , Quimiocinas/sangre , Femenino , Replicación ViralRESUMEN
In vitro studies have shown that deletion of nef and deleterious mutation in the Nef dimerization interface attenuates HIV replication and associated pathogenesis. Humanized rodents with human immune cells and lymphoid tissues are robust in vivo models for investigating the interactions between HIV and the human immune system. Here, we demonstrate that nef deletion impairs HIV replication and HIV-induced immune dysregulation in the blood and human secondary lymphoid tissue (human spleen) in bone marrow-liver-thymus-spleen (BLTS) humanized mice. Furthermore, we also show that nef defects (via deleterious mutations in the dimerization interface) impair HIV replication and HIV-induced immune dysregulation in the blood and human spleen in BLTS-humanized mice. We demonstrate that the reduced replication of nef-deleted and nef-defective HIV is associated with robust antiviral innate immune response, and T helper 1 response. Our results support the proposition that Nef may be a therapeutic target for adjuvants in HIV cure strategies.
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Modelos Animales de Enfermedad , Infecciones por VIH , VIH-1 , Hígado , Bazo , Viremia , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Ratones , Humanos , Viremia/inmunología , Bazo/inmunología , Bazo/virología , VIH-1/inmunología , VIH-1/genética , VIH-1/fisiología , Hígado/virología , Hígado/inmunología , Hígado/patología , Médula Ósea/virología , Médula Ósea/inmunología , Timo/inmunología , Timo/virología , Inmunidad InnataRESUMEN
Fulminant viral hepatitis (FH) represents a significant clinical challenge, with its pathogenesis not yet fully elucidated. Heat shock protein (HSP)70, a molecular chaperone protein with a broad range of cytoprotective functions, is upregulated in response to stress. However, the role of HSP70 in FH remains to be investigated. Notably, HSP70 expression is upregulated in the livers of coronavirus-infected mice and patients. Therefore, we investigated the mechanistic role of HSP70 in coronavirus-associated FH pathogenesis. FH was induced in HSP70-deficient (HSP70 KO) mice or in WT mice treated with the HSP70 inhibitor VER155008 when infected with the mouse hepatitis virus strain A59 (MHV-A59). MHV-A59-infected HSP70 KO mice exhibited significantly reduced liver damage and mortality. This effect was attributed to decreased infiltration of monocyte-macrophages and neutrophils in the liver of HSP70 KO mice, resulting in lower levels of inflammatory cytokines such as IL-1ß, TNFα, and IL-6, and a reduced viral load. Moreover, treatment with the HSP70 inhibitor VER155008 protected mice from MHV-A59-induced liver damage and FH mortality. In summary, HSP70 promotes coronavirus-induced FH pathogenesis by enhancing the infiltration of monocyte-macrophages and neutrophils and promoting the secretion of inflammatory cytokines. Therefore, HSP70 is a potential therapeutic target in viral FH intervention.
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Proteínas HSP70 de Choque Térmico , Hígado , Ratones Endogámicos C57BL , Ratones Noqueados , Virus de la Hepatitis Murina , Animales , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Virus de la Hepatitis Murina/patogenicidad , Ratones , Hígado/patología , Hígado/virología , Hígado/metabolismo , Citocinas/metabolismo , Humanos , Hepatitis Viral Animal/inmunología , Hepatitis Viral Animal/patología , Hepatitis Viral Animal/virología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Masculino , Macrófagos/inmunología , Nucleósidos de PurinaRESUMEN
Hepatitis B Virus (HBV) is a stealthy and insidious pathogen capable of inducing chronic necro-inflammatory liver disease and hepatocellular carcinoma (HCC), resulting in over one million deaths worldwide per year. The traditional understanding of Chronic Hepatitis B (CHB) progression has focused on the complex interplay among ongoing virus replication, aberrant immune responses, and liver pathogenesis. However, the dynamic progression and crucial factors involved in the transition from HBV infection to immune activation and intrahepatic inflammation remain elusive. Recent insights have illuminated HBV's exploitation of the sodium taurocholate co-transporting polypeptide (NTCP) and manipulation of the cholesterol transport system shared between macrophages and hepatocytes for viral entry. These discoveries deepen our understanding of HBV as a virus that hijacks hepatocyte metabolism. Moreover, hepatic niche macrophages exhibit significant phenotypic and functional diversity, zonal characteristics, and play essential roles, either in maintaining liver homeostasis or contributing to the pathogenesis of chronic liver diseases. Therefore, we underscore recent revelations concerning the importance of hepatic niche macrophages in the context of viral hepatitis. This review particularly emphasizes the significant role of HBV-induced metabolic changes in hepatic macrophages as a key factor in the transition from viral infection to immune activation, ultimately culminating in liver inflammation. These metabolic alterations in hepatic macrophages offer promising targets for therapeutic interventions and serve as valuable early warning indicators, shedding light on the disease progression.
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Virus de la Hepatitis B , Hepatitis B Crónica , Hígado , Macrófagos , Humanos , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/fisiología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Animales , Hígado/inmunología , Hígado/virología , Hígado/metabolismo , Hígado/patología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/virología , Inflamación/inmunología , Inflamación/metabolismo , Hepatocitos/metabolismo , Hepatocitos/inmunología , Hepatocitos/virologíaRESUMEN
Coronavirus disease 2019 (COVID-19), caused by the highly pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), primarily impacts the respiratory tract and can lead to severe outcomes such as acute respiratory distress syndrome, multiple organ failure, and death. Despite extensive studies on the pathogenicity of SARS-CoV-2, its impact on the hepatobiliary system remains unclear. While liver injury is commonly indicated by reduced albumin and elevated bilirubin and transaminase levels, the exact source of this damage is not fully understood. Proposed mechanisms for injury include direct cytotoxicity, collateral damage from inflammation, drug-induced liver injury, and ischemia/hypoxia. However, evidence often relies on blood tests with liver enzyme abnormalities. In this comprehensive review, we focused solely on the different histopathological manifestations of liver injury in COVID-19 patients, drawing from liver biopsies, complete autopsies, and in vitro liver analyses. We present evidence of the direct impact of SARS-CoV-2 on the liver, substantiated by in vitro observations of viral entry mechanisms and the actual presence of viral particles in liver samples resulting in a variety of cellular changes, including mitochondrial swelling, endoplasmic reticulum dilatation, and hepatocyte apoptosis. Additionally, we describe the diverse liver pathology observed during COVID-19 infection, encompassing necrosis, steatosis, cholestasis, and lobular inflammation. We also discuss the emergence of long-term complications, notably COVID-19-related secondary sclerosing cholangitis. Recognizing the histopathological liver changes occurring during COVID-19 infection is pivotal for improving patient recovery and guiding decision-making.
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COVID-19 , Hígado , SARS-CoV-2 , Humanos , COVID-19/complicaciones , COVID-19/patología , COVID-19/virología , Hígado/patología , Hígado/virología , SARS-CoV-2/patogenicidad , Hepatopatías/patología , Hepatopatías/virología , Hepatopatías/etiología , Hepatocitos/patología , Hepatocitos/virologíaRESUMEN
Chronic hepatitis C virus infection (HCV) causes liver inflammation and fibrosis, leading to the development of severe liver disease, such as cirrhosis or hepatocellular carcinoma (HCC). Approval of direct-acting antiviral drug combinations has revolutionized chronic HCV therapy, with virus eradication in >98% of the treated patients. The efficacy of these treatments is such that it is formally possible for cured patients to carry formerly infected cells that display irreversible transcriptional alterations directly caused by chronic HCV Infection. Combining differential transcriptomes from two different persistent infection models, we observed a major reversion of infection-related transcripts after complete infection elimination. However, a small number of transcripts were abnormally expressed in formerly infected cells. Comparison of the results obtained in proliferating and growth-arrested cell culture models suggest that permanent transcriptional alterations may be established by several mechanisms. Interestingly, some of these alterations were also observed in the liver biopsies of virologically cured patients. Overall, our data suggest a direct and permanent impact of persistent HCV infection on the host cell transcriptome even after virus elimination, possibly contributing to the development of HCC.
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Antivirales , Hepacivirus , Hepatitis C Crónica , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , Hepacivirus/genética , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virología , Transcriptoma , Infección Persistente/virología , Perfilación de la Expresión Génica , Hígado/virología , Hígado/patología , Carcinoma Hepatocelular/virología , Transcripción Genética/efectos de los fármacosRESUMEN
Risk factors for hepatocarcinogenesis include chronic inflammation due to viral infection, liver fibrosis, and aging. In this study, we separated carcinogenic and non-carcinogenic cases due to hepatitis C virus (HCV) infection, aiming to comprehensively analyze miRNA expression in liver tissues by age, and identify factors that contribute to carcinogenesis. Total RNA was extracted from 360 chronic hepatitis C (CH), 43 HCV infected hepatocellular carcinoma (HCC), and surrounding non-tumor (SNT) tissues. MicroRNA (miRNA) expression patterns were analyzed using microarray. Using machine learning, we extracted characteristic miRNA expression patterns for each disease and age. There were no age-dependent changes in miRNA expression in the disease-specific comparisons; however, miRNA expression differed among the age groups of 50, 60, and 70 years of age between CH and SNT. The expression of miRNA was different between SNT and HCC only in patients in their 70s. Of the 55 miRNAs with significant differences in expression between CH and SNT, 34 miRNAs showed significant differences in expression even in the degree of liver fibrosis. The observation that miRNAs involved in hepatocarcinogenesis differ at different ages suggests that the mechanisms of carcinogenesis differ by age group as well. We also found that many miRNAs whose expression did not affect liver fibrosis were involved in carcinogenesis. These findings are expected to define biomarkers for detection of HCC at early stage, and develop novel therapeutic targets for HCC.
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Carcinoma Hepatocelular , Hepatitis C Crónica , Neoplasias Hepáticas , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Persona de Mediana Edad , Masculino , Femenino , Anciano , Hepatitis C Crónica/genética , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/patología , Cirrosis Hepática/genética , Cirrosis Hepática/virología , Cirrosis Hepática/patología , Hígado/metabolismo , Hígado/patología , Hígado/virología , Perfilación de la Expresión Génica , Carcinogénesis/genética , Adulto , Regulación Neoplásica de la Expresión GénicaRESUMEN
HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.
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Southern Blotting , ADN Circular , ADN Viral , Virus de la Hepatitis B , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Humanos , ADN Viral/genética , ADN Viral/aislamiento & purificación , ADN Circular/aislamiento & purificación , ADN Circular/análisis , ADN Circular/genética , Southern Blotting/métodos , Hepatitis B/virología , Hepatitis B/diagnóstico , Hígado/virologíaRESUMEN
Hepatitis B virus (HBV) developed highly intricates mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. With the aid of a series of classical analytical methods such as ultrafiltration, and Southern and Northern blots, a general framework of HBV life cycle has been established. However, this picture still lacks many key histological contexts which involves pathophysiological changes of hepatocytes, non-parenchymal cells, infiltrated leukocytes, and associated extracellular matrix. Here, we describe a CISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in liver tissue of chronic hepatitis B patients. By coupling it with immunohistochemistry and other histological stains, much richer information regarding the HBV-induced pathological changes can be harvested.
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ADN Viral , Virus de la Hepatitis B , Hibridación in Situ , Hígado , ARN Viral , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Humanos , Hibridación in Situ/métodos , Hígado/virología , Hígado/metabolismo , ADN Viral/genética , ARN Viral/genética , Hepatitis B Crónica/virología , Compuestos Cromogénicos , Inmunohistoquímica/métodos , ADN Circular/genética , ADN Circular/análisisRESUMEN
Chimeric mouse models with a humanized liver (Hu-HEP mice) provide a unique tool to study human hepatotropic virus diseases, including viral infection, viral pathogenesis, and anti-viral therapy. Here, we describe a detailed protocol for studying hepatitis B infection in NRG-derived fumarylacetoacetate hydrolase (FAH) knockout mice repopulated with human hepatocytes (FRG-Hu HEP mice). The procedures include (1) maintenance and genotyping of the FRG mice, (2) intrasplenic injection of primary human hepatocytes (PHH), (3) 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) drug reduction cycling to improve human hepatocyte repopulation, (4) human albumin detection, and (5) HBV infection and detection. The method is simple and allows for highly reproducible generation of FRG-Hu HEP mice for HBV infection and therapy investigations.
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Modelos Animales de Enfermedad , Virus de la Hepatitis B , Hepatitis B , Hepatocitos , Hidrolasas , Hígado , Ratones Noqueados , Animales , Humanos , Ratones , Hidrolasas/genética , Hidrolasas/metabolismo , Hidrolasas/deficiencia , Hepatitis B/virología , Virus de la Hepatitis B/genética , Hígado/virología , Hígado/patología , Hepatocitos/virología , Hepatocitos/trasplante , Ratones Endogámicos NOD , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Quimera , Ciclohexanonas , NitrobenzoatosRESUMEN
The hepatitis delta virus (HDV) is a small RNA virus (1700 base pairs), which uses the surface proteins of the hepatitis B virus (HBV) as an envelope. Accurate and reliable quantitative detection of HDV RNA is central for scientific and translational clinical research or diagnostic purposes. However, HDV poses challenges for nucleic acid amplification techniques: (1) the circular genome displays high intramolecular base pairing; (2) high content of cytosine and guanine; and (3) enormous genomic diversity among the eight known HDV genotypes (GTs). Here, we provide step-by-step instructions for (A) a manual workflow to perform a quantitative HDV reverse transcription (RT)-PCR from serum and liver tissue and (B) a quantitative HDV RT-PCR assay with whole process control to be used for serum or plasma samples run on a fully automated system. Both assays target the conserved ribozyme region and demonstrate inclusivity for all eight HDV GTs. The choice of assay depends on the experimental needs and equipment availability. While the former is ideal for scientific research laboratories, the latter provides a useful tool in the field of translational research or diagnostics.
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Hepatitis D , Virus de la Hepatitis Delta , Hígado , ARN Viral , Flujo de Trabajo , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/aislamiento & purificación , Humanos , ARN Viral/genética , Hepatitis D/diagnóstico , Hepatitis D/virología , Hígado/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , GenotipoRESUMEN
Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.
Asunto(s)
Linfocitos T CD8-positivos , Hepatitis B Crónica , Hígado , Animales , Humanos , Masculino , Ratones , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/inmunología , Hepatocitos/virología , Hígado/inmunología , Hígado/virología , Fosforilación , Transducción de Señal , Activación de LinfocitosRESUMEN
Hepatic injuries in COVID-19 are not yet fully understood and indirect pathways (without viral replication in the liver) have been associated with the activation of vascular mechanisms of liver injury in humans infected with SARS-CoV-2. Golden Syrian hamsters are an effective model for experimental reproduction of moderate and self-limiting lung disease during SARS-CoV-2 infection. As observed in humans, this experimental model reproduces lesions of bronchointerstitial pneumonia and pulmonary vascular lesions, including endotheliitis (attachment of lymphoid cells to the luminal surface of endothelium). Extrapulmonary vascular lesions are well documented in COVID-19, but such extrapulmonary vascular lesions have not yet been described in the Golden Syrian hamster model of SARS-CoV-2 infection. The study aimed to evaluate microscopic liver lesions in Golden Syrian hamsters experimentally infected with SARS-CoV-2. In total, 38 conventional Golden Syrian hamsters, divided into infected group (n=24) and mock-infected group (n=14), were euthanized at 2-, 3-, 4-, 5-, 7-, 14-, and 15-days post infection with SARS-CoV-2. Liver fragments were evaluated by histopathology and immunohistochemical detection of SARS-CoV-2 Spike S2 antigens. The frequencies of portal vein endotheliitis, lobular activity, hepatocellular degeneration, and lobular vascular changes were higher among SARS-CoV-2-infected animals. Spike S2 antigen was not detected in liver. The main results indicate that SARS-CoV-2 infection exacerbated vascular and inflammatory lesions in the liver of hamsters with pre-existing hepatitis of unknown origin. A potential application of this animal model in studies of the pathogenesis and evolution of liver lesions associated with SARS-CoV-2 infection still needs further evaluation.
Asunto(s)
COVID-19 , Modelos Animales de Enfermedad , Hígado , Mesocricetus , SARS-CoV-2 , Animales , COVID-19/patología , Cricetinae , Hígado/patología , Hígado/virología , MasculinoRESUMEN
Chronic Hepatitis B virus (CHB) infection is a global health challenge, causing damage ranging from hepatitis to cirrhosis and hepatocellular carcinoma. In our study, single-cell RNA sequencing (scRNA-seq) analysis was performed in livers from mice models with chronic inflammation induced by CHB infection and we found that endothelial cells (ECs) exhibited the largest number of differentially expressed genes (DEGs) among all ten cell types. NF-κB signaling was activated in ECs to induce cell dysfunction and subsequent hepatic inflammation, which might be mediated by the interaction of macrophage-derived and cholangiocyte-derived VISFATIN/Nampt signaling. Moreover, we divided ECs into three subclusters, including periportal ECs (EC_Z1), midzonal ECs (EC_Z2), and pericentral ECs (EC_Z3) according to hepatic zonation. Functional analysis suggested that pericentral ECs and midzonal ECs, instead of periportal ECs, were more vulnerable to HBV infection, as the VISFATIN/Nampt- NF-κB axis was mainly altered in these two subpopulations. Interestingly, pericentral ECs showed increasing communication with macrophages and cholangiocytes via the Nampt-Insr and Nampt-Itga5/Itgb1 axis upon CHB infection, which contribute to angiogenesis and vascular capillarization. Additionally, ECs, especially pericentral ECs, showed a close connection with nature killer (NK) cells and T cells via the Cxcl6-Cxcr6 axis, which is involved in shaping the microenvironment in CHB mice livers. Thus, our study described the heterogeneity and functional alterations of three subclusters in ECs. We revealed the potential role of VISFATIN/Nampt signaling in modulating ECs characteristics and related hepatic inflammation, and EC-derived chemokine Cxcl16 in shaping NK and T cell recruitment, providing key insights into the multifunctionality of ECs in CHB-associated pathologies.
Asunto(s)
Células Endoteliales , Hepatitis B Crónica , Análisis de la Célula Individual , Animales , Hepatitis B Crónica/virología , Hepatitis B Crónica/genética , Hepatitis B Crónica/metabolismo , Ratones , Células Endoteliales/metabolismo , Células Endoteliales/virología , Análisis de Secuencia de ARN , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Transducción de Señal , Hígado/metabolismo , Hígado/virología , Hígado/patología , FN-kappa B/metabolismo , Masculino , Nicotinamida Fosforribosiltransferasa/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , HumanosRESUMEN
The development of direct-acting antivirals (DAAs) against hepatitis C virus (HCV) has revolutionized the management of this pathology, as their use allows viral elimination in a large majority of patients. Nonetheless, HCV remains a major public health problem due to the multiple challenges associated with its diagnosis, treatment availability and development of a prophylactic vaccine. Moreover, HCV-cured patients still present an increased risk of developing hepatic complications such as hepatocellular carcinoma. In the present review, we aim to summarize the impact that HCV infection has on a wide variety of peripheral and intrahepatic cell populations, the alterations that remain following DAA treatment and the potential molecular mechanisms implicated in their long-term persistence. Finally, we consider how recent developments in single-cell multiomics could refine our understanding of this disease in each specific intrahepatic cell population and drive the field to explore new directions for the development of chemo-preventive strategies.
Asunto(s)
Antivirales , Hepacivirus , Humanos , Antivirales/uso terapéutico , Antivirales/farmacología , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Hígado/metabolismo , Hígado/virología , Hígado/patología , Hígado/efectos de los fármacos , Carcinoma Hepatocelular/virología , Carcinoma Hepatocelular/metabolismo , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virologíaRESUMEN
Background: Coronaviruses (CoVs) belong to the RNA viruses family. The viruses in this family are known to cause mild respiratory disease in humans. The origin of the novel SARS-COV2 virus that caused the coronavirus-19 disease (COVID-19) is the Wuhan city in China from where it disseminated to cause a global pandemic. Although lungs are the predominant target organ for Coronavirus Disease-19 (COVID-19), since its outbreak, the disease is known to affect heart, blood vessels, kidney, intestine, liver and brain. This review aimed to summarize the catastrophic impacts of Coronavirus disease-19 on heart and liver along with its mechanisms of pathogenesis. Methods: The information used in this review was obtained from relevant articles published on PubMed, Google Scholar, Google, WHO website, CDC and other sources. Key searching statements and phrases related to COVID-19 were used to retrieve information. Original research articles, review papers, research letters and case reports were used as a source of information. Results: Besides causing severe lung injury, COVID-19 has also been reported to affect and cause dysfunction of many other organs. COVID-19 infection can affect people by downregulating membrane-bound active angiotensin-converting enzyme (ACE). People who have deficient ACE2 expression are more vulnerable to COVID-19 infection. The patients' pre-existing co-morbidities are major risk factors that predispose individuals to severe COVID-19. Conclusion: The disease severity and its broad spectrum phenotype is a result of combined direct and indirect pathogenic factors. Therefore, protocols that harmonize many therapeutic preferences should be the best alternatives to de-escalate the disease and obviate deaths caused as a result of multiple organ damage and dysfunction induced by the disease.