RESUMO
Post-acute cardiac sequelae, following SARS-CoV-2 infection, are well recognized as complications of COVID-19. We have previously shown the persistence of autoantibodies against antigens in skin, muscle, and heart in individuals following severe COVID-19; the most common staining on skin tissue displayed an inter-cellular cement pattern consistent with antibodies against desmosomal proteins. Desmosomes play a critical role in maintaining the structural integrity of tissues. For this reason, we analyzed desmosomal protein levels and the presence of anti-desmoglein (DSG) 1, 2, and 3 antibodies in acute and convalescent sera from patients with COVID-19 of differing clinical severity. We find increased levels of DSG2 protein in sera from acute COVID-19 patients. Furthermore, we find that DSG2 autoantibody levels are increased significantly in convalescent sera following severe COVID-19 but not in hospitalized patients recovering from influenza infection or healthy controls. Levels of autoantibody in sera from patients with severe COVID-19 were comparable to levels in patients with non-COVID-19-associated cardiac disease, potentially identifying DSG2 autoantibodies as a novel biomarker for cardiac damage. To determine if there was any association between severe COVID-19 and DSG2, we stained post-mortem cardiac tissue from patients who died from COVID-19 infection. This confirmed DSG2 protein within the intercalated discs and disruption of the intercalated disc between cardiomyocytes in patients who died from COVID-19. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection.
Assuntos
Autoanticorpos , COVID-19 , Humanos , Autoanticorpos/metabolismo , Soroterapia para COVID-19 , SARS-CoV-2 , MiocárdioRESUMO
OBJECTIVE: Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described, little is known about the location, phenotype and function of CD4+ TRM. DESIGN: We used multiparametric flow cytometry, histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype, function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS: Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69-, CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation, phenotypical profile (CXCR6+CD49a+S1PR1-PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely, CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype, a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes, these cells also formed part of the long-term resident pool, persisting in HLA-mismatched allografts. Notably, frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally, we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS: High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population, respectively, both shaped by the liver microenvironment to achieve diverse immunosurveillance.
Assuntos
Linfócitos T CD4-Positivos , Fígado , Linfócitos T CD8-Positivos , Citocinas/imunologia , Humanos , Memória Imunológica , Fígado/imunologia , Monitorização ImunológicaRESUMO
BACKGROUND AND AIMS: Lifetime risk of biliary tract cancer (BTC) in primary sclerosing cholangitis (PSC) may exceed 20%, and BTC is currently the leading cause of death in patients with PSC. To open new avenues for management, we aimed to delineate clinically relevant genomic and pathological features of a large panel of PSC-associated BTC (PSC-BTC). APPROACH AND RESULTS: We analyzed formalin-fixed, paraffin-embedded tumor tissue from 186 patients with PSC-BTC from 11 centers in eight countries with all anatomical locations included. We performed tumor DNA sequencing at 42 clinically relevant genetic loci to detect mutations, translocations, and copy number variations, along with histomorphological and immunohistochemical characterization. Regardless of the anatomical localization, PSC-BTC exhibited a uniform molecular and histological characteristic similar to extrahepatic cholangiocarcinoma. We detected a high frequency of genomic alterations typical of extrahepatic cholangiocarcinoma, such as TP53 (35.5%), KRAS (28.0%), CDKN2A (14.5%), and SMAD4 (11.3%), as well as potentially druggable mutations (e.g., HER2/ERBB2). We found a high frequency of nontypical/nonductal histomorphological subtypes (55.2%) and of the usually rare BTC precursor lesion, intraductal papillary neoplasia (18.3%). CONCLUSIONS: Genomic alterations in PSC-BTC include a significant number of putative actionable therapeutic targets. Notably, PSC-BTC shows a distinct extrahepatic morpho-molecular phenotype, independent of the anatomical location of the tumor. These findings advance our understanding of PSC-associated cholangiocarcinogenesis and provide strong incentives for clinical trials to test genome-based personalized treatment strategies in PSC-BTC.
Assuntos
Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/genética , Colangite Esclerosante/complicações , Adolescente , Adulto , Idoso , Neoplasias dos Ductos Biliares/mortalidade , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/terapia , Criança , Colangiocarcinoma/mortalidade , Colangiocarcinoma/patologia , Colangiocarcinoma/terapia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Feminino , Genes p53 , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto JovemRESUMO
Increased use of high-risk allografts is critical to meet the demand for liver transplantation. We aimed to identify criteria predicting viability of organs, currently declined for clinical transplantation, using functional assessment during normothermic machine perfusion (NMP). Twelve discarded human livers were subjected to NMP following static cold storage. Livers were perfused with a packed red cell-based fluid at 37°C for 6 hours. Multilevel statistical models for repeated measures were employed to investigate the trend of perfusate blood gas profiles and vascular flow characteristics over time and the effect of lactate-clearing (LC) and non-lactate-clearing (non-LC) ability of the livers. The relationship of lactate clearance capability with bile production and histological and molecular findings were also examined. After 2 hours of perfusion, median lactate concentrations were 3.0 and 14.6 mmol/L in the LC and non-LC groups, respectively. LC livers produced more bile and maintained a stable perfusate pH and vascular flow >150 and 500 mL/minute through the hepatic artery and portal vein, respectively. Histology revealed discrepancies between subjectively discarded livers compared with objective findings. There were minimal morphological changes in the LC group, whereas non-LC livers often showed hepatocellular injury and reduced glycogen deposition. Adenosine triphosphate levels in the LC group increased compared with the non-LC livers. We propose composite viability criteria consisting of lactate clearance, pH maintenance, bile production, vascular flow patterns, and liver macroscopic appearance. These have been tested successfully in clinical transplantation. In conclusion, NMP allows an objective assessment of liver function that may reduce the risk and permit use of currently unused high-risk livers.
Assuntos
Transplante de Fígado/efeitos adversos , Preservação de Órgãos/normas , Traumatismo por Reperfusão/diagnóstico , Sobrevivência de Tecidos , Coleta de Tecidos e Órgãos/efeitos adversos , Adulto , Idoso , Estudos de Viabilidade , Feminino , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Preservação de Órgãos/métodos , Perfusão/métodos , Perfusão/normas , Prognóstico , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/prevenção & controleRESUMO
Ischemia/reperfusion injury (IRI) is the main cause of complications following liver transplantation. Reactive oxygen species (ROS) were thought to be the main regulators of IRI. However, recent studies demonstrate that ROS activate the cytoprotective mechanism of autophagy promoting cell survival. Liver IRI initially damages the liver endothelial cells (LEC), but whether ROS-autophagy promotes cell survival in LEC during IRI is not known. Primary human LEC were isolated from human liver tissue and exposed to an in vitro model of IRI to assess the role of autophagy in LEC. The role of autophagy during liver IRI in vivo was assessed using a murine model of partial liver IRI. During IRI, ROS specifically activate autophagy-related protein (ATG) 7 promoting autophagic flux and the formation of LC3B-positive puncta around mitochondria in primary human LEC. Inhibition of ROS reduces autophagic flux in LEC during IRI inducing necrosis. In addition, small interfering RNA knockdown of ATG7 sensitized LEC to necrosis during IRI. In vivo murine livers in uninjured liver lobes demonstrate autophagy within LEC that is reduced following IRI with concomitant reduction in autophagic flux and increased cell death. In conclusion, these findings demonstrate that during liver IRI ROS-dependent autophagy promotes the survival of LEC, and therapeutic targeting of this signaling pathway may reduce liver IRI following transplantation.
Assuntos
Células Endoteliais/fisiologia , Transplante de Fígado/efeitos adversos , Mitofagia/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/patologia , Animais , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Sobrevivência Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Fígado/citologia , Fígado/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/etiologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND & AIMS: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. METHODS: To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. RESULTS: HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. CONCLUSIONS: We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization. LAY SUMMARY: HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.
Assuntos
Doença Hepática Terminal , Hepacivirus , Hepatite C Crônica , Interferons/farmacologia , Fígado , Replicação Viral/efeitos dos fármacos , Adulto , Antivirais/farmacologia , Compartimento Celular/efeitos dos fármacos , Doença Hepática Terminal/etiologia , Doença Hepática Terminal/metabolismo , Doença Hepática Terminal/virologia , Feminino , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C Crônica/complicações , Hepatite C Crônica/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , RNA Viral/análise , Análise de Sequência de RNA/métodosRESUMO
BACKGROUND & AIMS: Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. METHODS: In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. RESULTS: HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. CONCLUSIONS: Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. LAY SUMMARY: Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.
Assuntos
Hepacivirus/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Receptores de Ácidos Lisofosfatídicos/fisiologia , Replicação Viral , Animais , Linhagem Celular , Hepatite C Crônica/complicações , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Hepáticas/etiologia , Camundongos , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Transdução de SinaisRESUMO
Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are immune-mediated biliary diseases that demonstrate prominent and restricted genetic association with human leukocyte antigen (HLA) alleles. In PBC, anti-mitochondrial antibodies (AMA) are specific and used as diagnostic biomarkers. PSC-relevant auto-antibodies remain controversial despite a distinct HLA association that mirrors archetypical auto-antigen driven disorders. Herein, we compared antibody-secreting B cells (ASCs) in PSC and PBC liver explants to determine if liver-infiltrating ASCs represent an opportune and novel source of disease-relevant auto-antibodies. Using enzymatic digestion and mechanical disruption, liver mononuclear cells (LIMCs) were isolated from fresh PSC and PBC explants and plasmablast (CD19+CD27+CD38hiCD138-) and plasma cell (CD19+CD27+CD38hiCD138+) ASCs were enumerated by flow cytometry. We observed 45-fold fewer plasma cells in PSC explants (n = 9) compared to PBC samples (n = 5, p < 0.01) and 10-fold fewer IgA-, IgG- and IgM-positive ASCs (p < 0.05). Liver-infiltrating ASCs from PSC and PBC explants were functional and produced similar concentrations of IgA, IgG and IgM following 2 weeks of culture. Antibody production by PBC ASCs (n = 3) was disease-specific as AMA to pyruvate dehydrogenase complex E2 subunit (PDC-E2) was detected by immunostaining, immunoblotting and ELISA. Antibody profiling of PSC supernatants (n = 9) using full-length recombinant human protein arrays (Cambridge Protein Arrays) revealed reactivities to nucleolar protein 3 (5/9) and hematopoietic cell-specific Lyn substrate 1 (3/9). Array analysis of PBC supernatants (n = 3) detected reactivities to PDC-E2 and hexokinase 1 (3/3). In conclusion, we detected unique frequencies of liver-infiltrating ASCs in PSC and PBC and in so doing, highlight a feasible approach for understanding disease-relevant antibodies in PSC.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Colangite Esclerosante/diagnóstico , Colangite Esclerosante/imunologia , Cirrose Hepática Biliar/diagnóstico , Cirrose Hepática Biliar/imunologia , Fenótipo , Adolescente , Adulto , Idoso , Formação de Anticorpos/imunologia , Antígenos CD20/metabolismo , Autoanticorpos/sangue , Autoimunidade , Linfócitos B/patologia , Biomarcadores , Colangite Esclerosante/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Imunofenotipagem , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologia , Plasmócitos/metabolismo , Adulto JovemRESUMO
INTRODUCTION: CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. OBJECTIVE: To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. DESIGN: CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment. RESULTS: Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-ß at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC. CONCLUSIONS: Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury.
Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/metabolismo , Fígado/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Actinas/análise , Indutores da Angiogênese/farmacologia , Animais , Antígenos CD/análise , Antígenos de Neoplasias/análise , Becaplermina , Tetracloreto de Carbono , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Doença Crônica , Colágeno/metabolismo , Desmina/análise , Fibrose , Expressão Gênica , Células Estreladas do Fígado/química , Humanos , Inflamação/genética , Fígado/química , Cirrose Hepática/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Vimentina/análiseRESUMO
BACKGROUND & AIMS: T-cell-mediated biliary injury is a feature of primary sclerosing cholangitis (PSC). We studied the roles of CD28(-) T cells in PSC and their regulation by vitamin D. METHODS: Peripheral and liver-infiltrating mononuclear cells were isolated from blood or fresh liver tissue. We analyzed numbers, phenotypes, functions, and localization patterns of CD28(-) T cells, along with their ability to activate biliary epithelial cells. We measured levels of tumor necrosis factor (TNF)α in liver tissues from patients with PSC and the effects of exposure to active vitamin D (1,25[OH]2D3) on expression of CD28. RESULTS: A significantly greater proportion of CD4(+) and CD8(+) T cells that infiltrated liver tissues of patients with PSC were CD28(-), compared with control liver tissue (CD4(+): 30.3% vs 2.5%, P < .0001; and CD8(+): 68.5% vs 31.9%, P < .05). The mean percentage of CD4(+)CD28(-) T cells in liver tissues from patients with PSC was significantly higher than from patients with primary biliary cirrhosis or nonalcoholic steatohepatitis (P < .05). CD28(-) T cells were activated CD69(+)CD45RA(-) C-C chemokine receptor (CCR)7(-) effector memory and perforin(+) granzyme B(+) cytotoxic cells, which express CD11a, CX3CR1, C-X3-C motif receptor 6 (CXCR6), and CCR10-consistent with their infiltration of liver and localization around bile ducts. Compared with CD28(+) T cells, activated CD28(-) T cells produced significantly higher levels of interferon γ and TNFα (P < .05), and induced up-regulation of intercellular cell adhesion molecule-1, HLA-DR, and CD40 by primary epithelial cells (3.6-fold, 1.5-fold, and 1.2-fold, respectively). Liver tissue from patients with PSC contained high levels of TNFα; TNFα down-regulated the expression of CD28 by T cells in vitro (P < .01); this effect was prevented by administration of 1,25(OH)2D3 (P < .05). CONCLUSIONS: Inflammatory CD28(-) T cells accumulate in livers of patients with PSC and localize around bile ducts. The TNFα-rich microenvironment of this tissue promotes inflammation; these effects are reversed by vitamin D in vitro.
Assuntos
Antígenos CD28/deficiência , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Colangite Esclerosante/etiologia , Fígado/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Citocinas/metabolismo , Humanos , Técnicas In Vitro , Interferon gama/metabolismo , Fígado/patologia , Fator de Necrose Tumoral alfa/metabolismo , Vitamina D/farmacologiaRESUMO
Obesity is increasingly prevalent, strongly associated with nonalcoholic liver disease, and a risk factor for numerous cancers. Here, we describe the liver-related consequences of long-term diet-induced obesity. Mice were exposed to an extended obesity model comprising a diet high in trans-fats and fructose corn syrup concurrent with a sedentary lifestyle. Livers were assessed histologically using the nonalcoholic fatty liver disease (NAFLD) activity score (Kleiner system). Mice in the American Lifestyle-Induced Obesity Syndrome (ALIOS) model developed features of early nonalcoholic steatohepatitis at 6 months (mean NAFLD activity score = 2.4) and features of more advanced nonalcoholic steatohepatitis at 12 months, including liver inflammation and bridging fibrosis (mean NAFLD activity score = 5.0). Hepatic expression of lipid metabolism and insulin signaling genes were increased in ALIOS mice compared with normal chow-fed mice. Progressive activation of the mouse hepatic stem cell niche in response to ALIOS correlated with steatosis, fibrosis, and inflammation. Hepatocellular neoplasms were observed in 6 of 10 ALIOS mice after 12 months. Tumors displayed cytological atypia, absence of biliary epithelia, loss of reticulin, alteration of normal perivenular glutamine synthetase staining (absent or diffuse), and variable α-fetoprotein expression. Notably, perivascular tumor cells expressed hepatic stem cell markers. These studies indicate an adipogenic lifestyle alone is sufficient for the development of nonalcoholic steatohepatitis, hepatic stem cell activation, and hepatocarcinogenesis in wild-type mice.
Assuntos
Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/patologia , Dieta Hiperlipídica/efeitos adversos , Frutose/efeitos adversos , Neoplasias Hepáticas/patologia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Carcinoma Hepatocelular/irrigação sanguínea , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/lesões , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/complicações , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Obesidade/complicações , Obesidade/patologia , Fatores de Transcrição SOX9/metabolismo , Comportamento Sedentário , Transdução de Sinais/genética , Células-Tronco/patologiaRESUMO
The relevance of claudin-6 and claudin-9 in hepatitis C virus (HCV) entry remains elusive. We produced claudin-6- or claudin-9-specific monoclonal antibodies that inhibit HCV entry into nonhepatic cells expressing exogenous claudin-6 or claudin-9. These antibodies had no effect on HCV infection of hepatoma cells or primary hepatocytes. Thus, although claudin-6 and claudin-9 can serve as entry factors in cell lines, HCV infection into human hepatocytes is not dependent on claudin-6 and claudin-9.
Assuntos
Claudinas/metabolismo , Hepacivirus/fisiologia , Hepatócitos/virologia , Internalização do Vírus , Anticorpos Monoclonais/imunologia , Células Cultivadas , HumanosRESUMO
The presence of CD8+ T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8+ T cell invasion into BEC. CD8+ T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8+ T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8+ T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin+ CD8+ T cells formed ß-catenin-associated interactions with BEC, were larger than E-cadherin- CD8+ T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin+ CD103+ CD69+ CD8+ T cells into BEC.
Assuntos
Ductos Biliares , Cirrose Hepática Biliar , Humanos , Ductos Biliares/metabolismo , Cirrose Hepática Biliar/patologia , Linfócitos T CD8-Positivos/metabolismo , Células Epiteliais/metabolismo , Caderinas/metabolismoRESUMO
BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS. METHODS: We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication. RESULTS: Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis. CONCLUSIONS: Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.
Assuntos
Barreira Hematoencefálica/virologia , Células Endoteliais/virologia , Hepacivirus/patogenicidade , Hepatite C/virologia , Microvasos/virologia , Adulto , Antivirais/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Permeabilidade Capilar , Estudos de Casos e Controles , Linhagem Celular Tumoral , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Células HEK293 , Hepacivirus/genética , Hepatite C/complicações , Hepatite C/mortalidade , Humanos , Imuno-Histoquímica , Fígado/virologia , Masculino , Microscopia Confocal , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Microvasos/patologia , Pessoa de Meia-Idade , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Virais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Internalização do Vírus , Replicação ViralRESUMO
UNLABELLED: B cells are present within chronically inflamed liver tissue and recent evidence implicates them in the progression of liver disease. In addition, a large proportion of hepatic lymphomas are of B-cell origin. The molecular signals that regulate normal and malignant B-cell recruitment into peripheral tissue from blood are poorly understood, leading us to study human B-cell migration through hepatic sinusoidal endothelial cells in flow-based adhesion assays. In such assays, human blood-derived B cells were captured from shear flow without a previous rolling phase and underwent firm adhesion mediated by vascular cell adhesion molecule-1 (VCAM-1). Unlike T cells, which displayed vigorous crawling behavior on the endothelium, B cells remained static before a proportion underwent transendothelial migration mediated by a combination of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion protein-1, common lymphatic endothelial and vascular endothelial receptor-1/stabilin-1, and the chemokine receptors, CXCR3 and CXCR4. B-cell lymphoma cell lines and primary malignant B cells from patients with chronic lymphocytic leukemia and marginal zone B cell lymphoma also underwent integrin-mediated firm adhesion involving ICAM-1 and/or VCAM-1 and demonstrated ICAM-1-dependent shape-change and crawling behavior. Unlike primary lymphocytes, the malignant cells did not undergo transendothelial migration, which could explain why lymphomas are frequently characterized by the intravascular accumulation of malignant cells in the hepatic sinusoids. CONCLUSION: Our findings demonstrate that distinct combinations of signals promote B-cell recruitment to the liver, suggesting the possibility of novel targets to modulate liver inflammation in disease. Certain features of lymphocyte homing are maintained in lymphoma recruitment to the liver, suggesting that therapeutic targets for lymphocyte recruitment may also prevent hepatic lymphoma dissemination.
Assuntos
Linfócitos B/metabolismo , Células Cultivadas/citologia , Células Endoteliais/citologia , Hepatócitos/metabolismo , Linfócitos B/citologia , Adesão Celular/fisiologia , Comunicação Celular/genética , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Células Cultivadas/fisiologia , Células Endoteliais/metabolismo , Feminino , Citometria de Fluxo , Hepatócitos/fisiologia , Humanos , Imuno-Histoquímica , Fígado/citologia , Fígado/metabolismo , Linfoma de Células B/patologia , Masculino , Valores de Referência , Estudos de AmostragemRESUMO
The 2022 worldwide epidemic of acute hepatitis and liver failure in young children has led to a focus on unusual causes for childhood acute hepatitis. In the UK epidemic, human herpes virus subtype 6B (HHV-6B) was detected along with adenovirus subtype-41F in severely affected children, especially in those requiring liver transplantation (LT). The lifting of COVID lock-down measures has coincided with the rise in these common childhood infections with a higher than expected rate of systemic complications. The sudden exposure of young children to common childhood infections from which they were protected during the pandemic may have induced an abnormal immune mediated response potentiated by multiple pathogen exposure. Primary HHV-6 infection is one such common childhood infection. Classically known as Roseola infantum due to the appearance of a widespread erythematous rash on fever subsidence (exanthema subitem), it has a peak incidence of 6-12 months of age and almost all children will have been infected by age 2. It is the virus most frequently associated with febrile convulsions but the more serious complications of hepatitis and liver failure are rare. We report on the historic cases of three female infants who had suspected primary HHV-6B infection, acute hepatitis and rapid progression to acute liver failure (ALF) requiring LT. Appearances of their native liver were identical to those described in children in the recent hepatitis epidemic. Deteriorating clinical trajectories of recurrent graft hepatitis and rejection-like episodes followed and all three succumbed to graft failure with HHV-6B detected posthumously in their liver allografts. Our case series and the serious complications observed with the recent rise in common childhood infections is a reminder that these routinely encountered pathogens can be deadly especially in the young immunologically untrained. We advocate for HHV-6 to be screened for routinely in children with acute hepatitis and the use of effective HHV-6 anti-viral prophylaxis to prevent recurrence post-transplant.
RESUMO
BACKGROUND: We aimed to predict response to biologics in inflammatory bowel disease (IBD) using computerized image analysis of probe confocal laser endomicroscopy (pCLE) in vivo and assess the binding of fluorescent-labeled biologics ex vivo. Additionally, we investigated genes predictive of anti-tumor necrosis factor (TNF) response. METHODS: Twenty-nine patients (15 with Crohn's disease [CD], 14 with ulcerative colitis [UC]) underwent colonoscopy with pCLE before and 12 to 14 weeks after starting anti-TNF or anti-integrin α4ß7 therapy. Biopsies were taken for fluorescein isothiocyanate-labeled infliximab and vedolizumab staining and gene expression analysis. Computer-aided quantitative image analysis of pCLE was performed. Differentially expressed genes predictive of response were determined and validated in a public cohort. RESULTS: In vivo, vessel tortuosity, crypt morphology, and fluorescein leakage predicted response in UC (area under the receiver-operating characteristic curve [AUROC], 0.93; accuracy 85%, positive predictive value [PPV] 89%; negative predictive value [NPV] 75%) and CD (AUROC, 0.79; accuracy 80%; PPV 75%; NPV 83%) patients. Ex vivo, increased binding of labeled biologic at baseline predicted response in UC (UC) (AUROC, 83%; accuracy 77%; PPV 89%; NPV 50%) but not in Crohn's disease (AUROC 58%). A total of 325 differentially expressed genes distinguished responders from nonresponders, 86 of which fell within the most enriched pathways. A panel including ACTN1, CXCL6, LAMA4, EMILIN1, CRIP2, CXCL13, and MAPKAPK2 showed good prediction of anti-TNF response (AUROC >0.7). CONCLUSIONS: Higher mucosal binding of the drug target is associated with response to therapy in UC. In vivo, mucosal and microvascular changes detected by pCLE are associated with response to biologics in inflammatory bowel disease. Anti-TNF-responsive UC patients have a less inflamed and fibrotic state pretreatment. Chemotactic pathways involving CXCL6 or CXCL13 may be novel targets for therapy in nonresponders.
Assuntos
Produtos Biológicos , Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Humanos , Doença de Crohn/diagnóstico por imagem , Doença de Crohn/tratamento farmacológico , Doença de Crohn/genética , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Doenças Inflamatórias Intestinais/diagnóstico por imagem , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/genética , Colite Ulcerativa/diagnóstico por imagem , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/genética , Fator de Necrose Tumoral alfa/uso terapêutico , Terapia Biológica , Produtos Biológicos/uso terapêutico , Expressão Gênica , Fluoresceínas/uso terapêutico , Lasers , Proteínas Adaptadoras de Transdução de Sinal , Proteínas com Domínio LIMRESUMO
BACKGROUND & AIMS: IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. METHODS: The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. RESULTS: IL-17(+) T cells comprised 1-3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on ß1 and ß2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17(+) cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. CONCLUSIONS: CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.
Assuntos
Hepatite/patologia , Hepatopatias/patologia , Receptores CCR6/metabolismo , Receptores CXCR3/metabolismo , Células Th17/patologia , Animais , Sistema Biliar/metabolismo , Sistema Biliar/patologia , Movimento Celular/fisiologia , Células Cultivadas , Quimiocina CCL20/metabolismo , Modelos Animais de Doenças , Hepatite/metabolismo , Humanos , Hepatopatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Células Th17/metabolismoRESUMO
BACKGROUND & AIMS: Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC. METHODS: We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion. RESULTS: HCV glycoproteins perturb tight and adherens junction protein expression, and increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-ß). Neutralization of both growth factors shows different roles for VEGF and TGFß in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle. CONCLUSIONS: These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Movimento Celular/fisiologia , Hepacivirus/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Hepáticas/fisiopatologia , Replicação Viral/fisiologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Polaridade Celular/fisiologia , Progressão da Doença , Glicoproteínas/fisiologia , Hepatite C/patologia , Hepatite C/fisiopatologia , Humanos , Neoplasias Hepáticas/patologia , Junções Íntimas/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
UNLABELLED: Primary sclerosing cholangitis (PSC) and autoimmune hepatitis are hepatic complications associated with inflammatory bowel disease (IBD). The expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) on mucosal endothelium is a prerequisite for the development of IBD, and it is also detected on the hepatic vessels of patients with liver diseases associated with IBD. This aberrant hepatic expression of MAdCAM-1 results in the recruitment of effector cells initially activated in the gut to the liver, in which they drive liver injury. However, the factors responsible for the aberrant hepatic expression of MAdCAM-1 are not known. In this study, we show that deamination of methylamine (MA) by vascular adhesion protein 1 (VAP-1) [a semicarbazide-sensitive amine oxidase (SSAO) expressed in the human liver] in the presence of tumor necrosis factor α induces the expression of functional MAdCAM-1 in hepatic endothelial cells and in intact human liver tissue ex vivo. This is associated with increased adhesion of lymphocytes from patients with PSC to hepatic vessels. Feeding mice MA, a constituent of food and cigarette smoke found in portal blood, led to VAP-1/SSAO-dependent MAdCAM-1 expression in mucosal vessels in vivo. CONCLUSION: Activation of VAP-1/SSAO enzymatic activity by MA, a constituent of food and cigarette smoke, induces the expression of MAdCAM-1 in hepatic vessels and results in the enhanced recruitment of mucosal effector lymphocytes to the liver. This could be an important mechanism underlying the hepatic complications of IBD.