RESUMO
Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV)-induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated that blocking dynamin decreased the infectivity of RRV, whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV, whereas late endosome inhibition had no effect. After infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human patients with BA experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that use Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10 and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction.NEW & NOTEWORTHY In this study, we have determined that the presence of the "SRL" peptide on RRV alters its method of endocytosis and intracellular trafficking through viral binding to heat shock cognate 70 protein. This initiates an inflammatory pathway that stimulates the release of cytokines associated with biliary damage and obstruction.
Assuntos
Atresia Biliar , Proteínas do Capsídeo , Modelos Animais de Doenças , Endocitose , Infecções por Rotavirus , Rotavirus , Atresia Biliar/metabolismo , Atresia Biliar/virologia , Animais , Camundongos , Infecções por Rotavirus/metabolismo , Infecções por Rotavirus/virologia , Humanos , Proteínas do Capsídeo/metabolismo , Receptor 3 Toll-Like/metabolismo , Sítios de Ligação , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSC70/genética , Camundongos Knockout , NF-kappa B/metabolismo , Transdução de Sinais , Clatrina/metabolismo , Camundongos Endogâmicos C57BL , Quimiocina CXCL10RESUMO
Biliary atresia (BA) is an obstructive neonatal cholangiopathy leading to liver cirrhosis and end stage liver disease. A Kasai portoenterostomy may restore biliary drainage, but most patients ultimately require liver transplantation for survival. At diagnosis, immune cells within the liver of patients with BA demonstrate a T-helper 1 (Th1) inflammatory profile similar to rhesus rotavirus (RRV)-infected mice livers developing BA. The transcription factor Tbx21 (T-bet) is essential for induction of a Th1 immune response in both the adaptive and innate immune system. Here we used animals with targeted deletion of the T-bet gene to determine its role in the progression of BA. Infection of newborn T-bet knockout (KO) pups with RRV resulted in a decreased Th1 inflammatory chemokine/cytokine profile when compared to infected wild-type mice. Analysis of the mononuclear cells profile from T-bet KO mice revealed both a significant decrease in the total number of CD3, CD4, and CD8 T cells and their effector molecules granzyme A, perforin, and FasL. Even though the percentage of T-bet KO mice displaying symptoms of an obstructive cholangiopathy and overall mortality rate was not different compared to wild-type mice, the extrahepatic bile ducts of T-bet KO mice remained patent.
Assuntos
Atresia Biliar/genética , Fígado/metabolismo , Infecções por Rotavirus/genética , Proteínas com Domínio T/genética , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Atresia Biliar/patologia , Atresia Biliar/cirurgia , Atresia Biliar/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Humanos , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos Knockout , Rotavirus/patogenicidade , Infecções por Rotavirus/complicações , Infecções por Rotavirus/virologia , Células Th1/imunologia , Células Th1/metabolismoRESUMO
BACKGROUND AND AIMS: Biliary atresia (BA) is a devastating cholangiopathy of infancy. Upon diagnosis, surgical reconstruction by Kasai hepatoportoenterostomy (HPE) restores biliary drainage in a subset of patients, but most patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling that of human BA. High-mobility group box 1 (HMGB1) is an important member of the danger-associated molecular patterns capable of mediating inflammation during infection-associated responses. In this study, we investigated the role of HMGB1 in BA pathogenesis. APPROACH AND RESULTS: In cholangiocytes, RRV induced the expression and release of HMGB1 through the p38 mitogen-activated protein kinase signaling pathway, and inhibition of p38 blocked HMGB1 release. Treatment of cholangiocytes with ethyl pyruvate suppressed the release of HMGB1. Administration of glycyrrhizin in vivo decreased symptoms and increased survival in the murine model of BA. HMGB1 levels were measured in serum obtained from infants with BA enrolled in the PROBE and START studies conducted by the Childhood Liver Disease Research Network. High HMGB1 levels were found in a subset of patients at the time of HPE. These patients had higher bilirubin levels 3 months post-HPE and a lower survival of their native liver at 2 years. CONCLUSIONS: These results suggest that HMGB1 plays a role in virus induced BA pathogenesis and could be a target for therapeutic interventions in a subset of patients with BA and high HMGB1.
Assuntos
Atresia Biliar/patologia , Doença Hepática Terminal/epidemiologia , Proteína HMGB1/sangue , Proteína HMGB1/metabolismo , Infecções por Rotavirus/patologia , Animais , Animais Recém-Nascidos , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Ductos Biliares/cirurgia , Atresia Biliar/sangue , Atresia Biliar/cirurgia , Atresia Biliar/virologia , Bilirrubina/sangue , Biomarcadores/sangue , Linhagem Celular , Pré-Escolar , Chlorocebus aethiops , Modelos Animais de Doenças , Doença Hepática Terminal/patologia , Células Epiteliais , Humanos , Lactente , Recém-Nascido , Camundongos , Portoenterostomia Hepática , Medição de Risco , Fatores de Risco , Rotavirus/metabolismo , Rotavirus/patogenicidade , Infecções por Rotavirus/virologia , Resultado do TratamentoRESUMO
Biliary atresia (BA) is a neonatal liver disease characterized by progressive fibroinflammatory obliteration of both intrahepatic and extrahepatic bile ducts. The etiologies of BA remain largely unknown, but rotavirus infection has been implicated at least for a subset of patients, and this causal relation has been well demonstrated in mouse models. In this study, we aim to further consolidate this evidence in human biliary organoids. We obtained seven batches of human biliary organoids cultured from fetal liver, adult liver, and bile duct tissues. We found that these organoids are highly susceptible and support the full life cycle of rotavirus infection in three-dimensional culture. The robust infection triggers active virus-host interactions, including interferon-based host defense mechanisms and injury responses. We have observed direct cytopathogenesis in organoids upon rotavirus infection, which may partially recapitulate the development of BA. Importantly, we have demonstrated the efficacy of mycophenolic acid and interferon alpha but not ribavirin in inhibiting rotavirus in biliary organoids. Furthermore, neutralizing antibody targeting rotavirus VP7 protein effectively inhibits infection in organoids. Thus, we have substantiated the causal evidence of rotavirus inducing BA in humans and provided potential strategies to combat the disease.IMPORTANCE There is substantial evidence indicating the possible involvement of rotavirus in biliary atresia (BA) development, at least in a subset of patients, but concrete proof remains lacking. In a mouse model, it has been well demonstrated that rotavirus can infect the biliary epithelium to cause biliary inflammation and obstruction, representing the pathogenesis of BA in humans. By using recently developed organoids technology, we now have demonstrated that human biliary organoids are susceptible to rotavirus infection, and this provokes active virus-host interactions and causes severe cytopathogenesis. Thus, our model recapitulates some essential aspects of BA development. Furthermore, we have demonstrated that antiviral drugs and neutralizing antibodies are capable of counteracting the infection and BA-like morphological changes, suggesting their potential for mitigating BA in patients.
Assuntos
Atresia Biliar/patologia , Efeito Citopatogênico Viral , Organoides/patologia , Organoides/virologia , Infecções por Rotavirus/patologia , Anticorpos Neutralizantes/farmacologia , Antígenos Virais/imunologia , Antivirais/farmacologia , Atresia Biliar/virologia , Proteínas do Capsídeo/imunologia , Interações entre Hospedeiro e Microrganismos , Humanos , Interferon-alfa/farmacologia , Ácido Micofenólico/farmacologia , Organoides/efeitos dos fármacos , Ribavirina/farmacologiaRESUMO
Biliary atresia (BA) is a devastating fibro-inflammatory disease characterized by the obstruction of extrahepatic and intrahepatic bile ducts in infants that can have fatal consequences, when not treated in a timely manner. It is the most common indication of pediatric liver transplantation worldwide and the development of new therapies, to alleviate the need of surgical intervention, has been hindered due to its complexity and lack of understanding of the disease pathogenesis. For that reason, significant efforts have been made toward the development of experimental models and strategies to understand the etiology and disease mechanisms and to identify novel therapeutic targets. The only characterized model of BA, using a Rhesus Rotavirus Type A infection of newborn BALB/c mice, has enabled the identification of key cellular and molecular targets involved in epithelial injury and duct obstruction. However, the establishment of an unleashed chronic inflammation followed by a progressive pathological wound healing process remains poorly understood. Like T cells, macrophages can adopt different functional programs [pro-inflammatory (M1) and resolutive (M2) macrophages] and influence the surrounding cytokine environment and the cell response to injury. In this review, we provide an overview of the immunopathogenesis of BA, discuss the implication of innate immunity in the disease pathogenesis and highlight their suitability as therapeutic targets.
Assuntos
Atresia Biliar/imunologia , Atresia Biliar/patologia , Imunidade Inata/imunologia , Animais , Atresia Biliar/terapia , Atresia Biliar/virologia , Colestase/etiologia , Colestase/metabolismo , Citocinas , Modelos Animais de Doenças , Progressão da Doença , Humanos , Imunidade Humoral , Inflamação , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Rotavirus/patogenicidade , Infecções por RotavirusRESUMO
Biliary atresia is a devastating neonatal cholangiopathy that affects both extra- and intrahepatic bile ducts progressing to fibrosis and end-stage liver disease by 2 years of age. Despite re-establishment of biliary drainage following a Kasai portoenterostomy (surgical procedure), many infants develop fibrosis requiring liver transplant. In the murine model of biliary atresia, rhesus rotavirus infection of newborn pups results in a cholangiopathy paralleling human biliary atresia and is used to study mechanistic aspects of the disease. The infected mice displayed histopathological signs similar to human biliary atresia, with bile duct obstruction, bile duct proliferation, and liver inflammation with fibrosis.
Assuntos
Atresia Biliar/etiologia , Atresia Biliar/virologia , Colestase/etiologia , Colestase/virologia , Rotavirus/patogenicidade , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
AIM: Rotaviruses have been associated with biliary atresia. This study investigated whether the rotavirus vaccine, which was introduced to Korea in 2008, had an impact on the incidence of biliary atresia. METHODS: We identified all rotavirus infections (n = 436 826) and biliary atresia cases (n = 528) diagnosed from 2006 to 2015 from insurance and health databases. The annual and seasonal incidence of biliary atresia and rotavirus infection rates in neonates and children were calculated. The difference in the risk of biliary atresia between rotavirus-infected and non-infected neonates was analysed. RESULTS: The incidence of rotavirus infections was 20.6 versus 13.4 per 1000 cases before (2006-2008) and after (2009-2015) vaccine implementation. However, neonatal rotavirus infection rates did not decrease, with an incidence of 14.5 versus 14.8 per 1000 cases before and after vaccination. The biliary atresia incidence remained constant at 12.0 per 100 000 cases. Rotavirus infections in neonates were a risk factor for biliary atresia (odds ratio 3.14, 95% confidence interval 1.87-5.26). CONCLUSION: Rotavirus vaccination had no impact on the incidence of biliary atresia, possibly because the vaccination did not change the neonatal rotavirus infection rate through herd immunity. However, rotavirus infections in neonates were significantly associated with biliary atresia.
Assuntos
Atresia Biliar/epidemiologia , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus , Atresia Biliar/virologia , Pré-Escolar , Feminino , Humanos , Incidência , Lactente , Masculino , República da Coreia/epidemiologia , Infecções por Rotavirus/complicações , Infecções por Rotavirus/epidemiologia , Estações do AnoRESUMO
Vascular damage has been reported to contribute to atresia formation in several diseases including biliary atresia. This study focused on the extrahepatic biliary plexus in experimental biliary atresia. Newborn BALB/cAnNCrl-pups were infected with rhesus rotavirus within 24 hr after birth to induce experimental biliary atresia. The extrahepatic biliary plexus was examined by confocal microscopy on whole-mount preparations, scored by three independent researchers, and further evaluated at the subcellular level with transmission electron microscopy. Imaging results revealed a progressive destruction of the extrahepatic biliary vascular plexus in the course of experimental biliary atresia induced by rotavirus infection. Endothelial cell damage was already visible as cell swelling and necrosis in the first days after infection and a damaged microcirculation that rapidly deteriorated with progression of obliterative cholangiopathy, was observed in the infected mice as early as 72 hr after birth. In experimental biliary atresia, the destruction of the extrahepatic biliary vascular plexus starts already in the first days postinfection and clearly precedes the morphological symptoms of atresia. The deterioration of the vascular bed architecture continues with disease progression. Therefore, we conclude that the (ultra)structural changes in the extrahepatic biliary microvasculature occurring before the visible onset of atresia has a predictive diagnostic value and this impairment in blood supply to the extrahepatic bile duct may be an important contributing factor to the pathogenesis of acquired biliary atresia. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:818-824, 2019. © 2018 Wiley Periodicals, Inc.
Assuntos
Ductos Biliares Extra-Hepáticos/irrigação sanguínea , Atresia Biliar/patologia , Microvasos/patologia , Infecções por Rotavirus/patologia , Rotavirus/patogenicidade , Animais , Animais Recém-Nascidos , Ductos Biliares Extra-Hepáticos/patologia , Ductos Biliares Extra-Hepáticos/virologia , Atresia Biliar/virologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microvasos/ultraestrutura , Microvasos/virologia , Infecções por Rotavirus/virologiaRESUMO
AIM: To analyze the expression and function of the Notch signaling target gene Hes1 in a rhesus rotavirus-induced mouse biliary atresia model. METHODS: The morphologies of biliary epithelial cells in biliary atresia patients and in a mouse model were examined by immunohistochemical staining. Then, the differential expression of Notch signaling pathway-related molecules was investigated. Further, the effects of the siRNA-mediated inhibition of Hes1 expression were examined using a biliary epithelial cell 3D culture system. RESULTS: Both immature (EpCAM+) and mature (CK19+) biliary epithelial cells were detected in the livers of biliary atresia patients without a ductile structure and in the mouse model with a distorted bile duct structure. The hepatic expression of transcripts for most Notch signaling molecules were significantly reduced on day 7 but recovered to normal levels by day 14, except for the target molecule Hes1, which still exhibited lower mRNA and protein levels. Expression of the Hes1 transcriptional co-regulator, RBP-Jκ was also reduced. A 3D gel culture system promoted the maturation of immature biliary epithelial cells, with increased expression of CK19+ cells and the formation of a duct-like structure. The administration of Hes1 siRNA blocked this process. As a result, the cells remained in an immature state, and no duct-like structure was observed. CONCLUSION: Our data indicated that Hes1 might contribute to the maturation and the cellular structure organization of biliary epithelial cells, which provides new insight into understanding the pathology of biliary atresia.
Assuntos
Ductos Biliares/patologia , Atresia Biliar/patologia , Fatores de Transcrição HES-1/metabolismo , Animais , Ductos Biliares/citologia , Atresia Biliar/cirurgia , Atresia Biliar/virologia , Técnicas de Cultura de Células , Células Cultivadas , Cisto do Colédoco/patologia , Cisto do Colédoco/cirurgia , Modelos Animais de Doenças , Regulação para Baixo , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Fígado/citologia , Fígado/patologia , Fígado/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Notch/metabolismo , Rotavirus/patogenicidade , Transdução de Sinais , Fatores de Transcrição HES-1/genéticaRESUMO
PURPOSE: To identify genes potentially involved in the pathogenesis of bile duct obstruction in biliary atresia (BA). METHODS: We used rhesus rotavirus (RRV) Balb/c mouse BA model to study BA. Liver and serum samples were harvested from BA and normal control (NC) groups at 1, 3, 5, 7, 10 and 14 days postinoculation. Serum total bilirubin (STB) and conjugated bilirubin (CB) were measured. Livers of each group at day 7 were used for a genome-wide expression analysis. Expression of TLR7 signaling pathway in liver was measured by immunohistochemical staining and western blotting, including expression of TLR7, activation of phosphorylated IRF7 and secretion of IFN-ß, IL-1α and IL-6. Cell viability and survival rate after RRV infection were measured by using TLR7 knockdown human cholangiocarcinoma cell RBE. RESULTS: STB was significantly elevated from day 5 postinoculation and CB was from day 7 postinoculation, while CK19 (the biomarker of biliary epithelial cells) expression by western blotting was decreased. By microarray analysis of liver tissues at day 7 postinoculation, TLR7 signaling pathway was up-regulated in BA mice. Based on the results of microarray analysis, the protein expression of TLR7 in the liver tissues of BA groups were found to be up-regulated from day 5 comparing to respective NC groups, although it was increased as pups aged in NC groups. And the level of p-IRF7 and secretion of cytokines were also statistically significant in BA groups. In vitro, TLR7 knockdown cell line showed less cellular proliferation and more susceptible to RRV infection. CONCLUSION: By in vivo study, TLR7 signal pathway was up-regulated in BA group; by additional in vitro study, intact TLR7 signal pathway might have some protective abilities in BA pathogenesis.
Assuntos
Atresia Biliar/genética , Atresia Biliar/virologia , Fígado/virologia , Glicoproteínas de Membrana/genética , Infecções por Rotavirus/complicações , Receptor 7 Toll-Like/genética , Transcriptoma , Animais , Atresia Biliar/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Humanos , Fígado/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Rotavirus/isolamento & purificação , Transdução de Sinais , Receptor 7 Toll-Like/metabolismoRESUMO
Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice.IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.
Assuntos
Atresia Biliar/patologia , Proteínas do Capsídeo/genética , Proteínas Mutantes/genética , Mutação Puntual , Rotavirus/patogenicidade , Animais , Animais Recém-Nascidos , Atresia Biliar/virologia , Proteínas do Capsídeo/metabolismo , Modelos Animais de Doenças , Camundongos , Proteínas Mutantes/metabolismo , Genética Reversa , Rotavirus/genética , Tropismo Viral , Replicação ViralRESUMO
Biliary atresia (BA) is a neonatal biliary system disease closely associated with viral infection and bile duct inflammation. Silver nanoparticles (AgNps) have previously revealed antiviral and anti-inflammatory properties. In this study, we have investigated the effects of AgNps in the treatment of the Rhesus rotavirus inoculation induced BA in mice. The morphology, liver histopathology, clinical biochemistry examination, and inflammatory cells were analyzed in BA mice. Results indicated that AgNps could significantly increase the survival rate of BA mice, and reduce jaundice and weight lost and the liver enzymes and bilirubin metabolism clinical parameters were close to the normal levels. Diminished numbers of NK cells were observed by flow cytometry analysis and immunohistochemical staining. Furthermore, the viral load was reduced and transcripts for TGF-ß mRNA were augmented after AgNps treatment. Collectively, our results suggest that AgNps treatment has beneficial effects on the BA mouse model partially through upregulation of TGF-ß.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , Atresia Biliar/tratamento farmacológico , Nanopartículas Metálicas/uso terapêutico , Infecções por Rotavirus/tratamento farmacológico , Rotavirus/efeitos dos fármacos , Prata/uso terapêutico , Animais , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/patologia , Atresia Biliar/patologia , Atresia Biliar/virologia , Modelos Animais de Doenças , Feminino , Icterícia/tratamento farmacológico , Icterícia/patologia , Icterícia/virologia , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/virologia , Camundongos Endogâmicos BALB C , Rotavirus/isolamento & purificação , Infecções por Rotavirus/complicações , Infecções por Rotavirus/patologiaRESUMO
Biliary atresia (BA) is a neonatal obstructive cholangiopathy that progresses to end-stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that the RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Using rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains that cause obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4-derived peptide (TRTRVSRLY) significantly reduced the ability of RRV to bind and infect cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains that do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by small interfering RNAs reduced RRV's ability to infect cholangiocytes. This virus-cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV-injected mice. CONCLUSION: The tripeptide SRL on RRV VP4 binds to the cholangiocyte membrane protein Hsc70, defining a novel binding site governing VP4 attachment. Investigations are underway to determine the cellular response to this interaction to understand how it contributes to the pathogenesis of BA. (Hepatology 2017;65:1278-1292).
Assuntos
Atresia Biliar/genética , Proteínas do Capsídeo/genética , Colangite/genética , Rotavirus/patogenicidade , Animais , Animais Recém-Nascidos , Ductos Biliares/citologia , Atresia Biliar/virologia , Células Cultivadas , Colangite/virologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Macaca mulatta , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Rotavirus/genética , Infecções por Rotavirus/patologia , Infecções por Rotavirus/fisiopatologia , Ligação Viral , Replicação ViralRESUMO
The Rhesus rotavirus (RRV) induced murine model of biliary atresia (BA) is a useful tool in studying the pathogenesis of this neonatal biliary obstructive disease. In this model, the mitogen associated protein kinase pathway is involved in RRV infection of biliary epithelial cells (cholangiocytes). We hypothesized that extracellular signal-related kinase (ERK) phosphorylation is integral to calcium influx, allowing for viral replication within the cholangiocyte. Utilizing ERK and calcium inhibitors in immortalized cholangiocytes and BALB/c pups, we determined that ERK inhibition resulted in reduced viral yield and subsequent decreased symptomatology in mice. In vitro, the RRV VP6 protein induced ERK phosphorylation, leading to cellular calcium influx. Pre-treatment with an ERK inhibitor or Verapamil resulted in lower viral yields. We conclude that the pathogenesis of RRV-induced murine BA is dependent on the VP6 protein causing ERK phosphorylation and triggering calcium influx allowing replication in cholangiocytes.
Assuntos
Antígenos Virais/metabolismo , Atresia Biliar/virologia , Proteínas do Capsídeo/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Infecções por Rotavirus/enzimologia , Infecções por Rotavirus/virologia , Rotavirus/metabolismo , Animais , Antígenos Virais/genética , Atresia Biliar/enzimologia , Atresia Biliar/metabolismo , Transporte Biológico , Cálcio/metabolismo , Proteínas do Capsídeo/genética , Modelos Animais de Doenças , Humanos , Macaca mulatta , Camundongos Endogâmicos BALB C , Fosforilação , Rotavirus/genética , Infecções por Rotavirus/metabolismoRESUMO
OBJECTIVES: To explore the evidence for viral infections triggering human biliary atresia (BA) by reviewing archival original articles that analyzed human samples via polymerase chain reaction (PCR) experiments, considering the recent experimental trend of extensive use of rotaviral BA animal models. METHODS: A PubMed search retrieved original articles that reported the results of PCR experiments for detecting viral DNA or RNA in patient samples as proof of past infection. Search terms included the often-debated DNA or RNA viruses and BA. Special focus was directed toward PCR analyses that targeted reovirus and rotavirus, where PCR accuracy, specimen characteristics and their interpretations were compared. RESULTS: Nineteen studies were conducted on 16 different kinds of viruses using PCR, with 5 studies on reovirus, 3 on rotavirus, 10 on cytomegalovirus, 5 on Epstein-Barr virus, 4 on parvovirus B19, and so on. Among the papers suggesting a possible viral link to only BA, there was no study on reovirus, 1 on rotavirus, 3 on cytomegalovirus, 1 on EB virus, and 1 on papillomavirus. Of the 6 PCR studies on Reoviridae, 3 on reovirus and 2 on rotavirus were evaluated rigorously for experimental accuracy, including their sensitivity. Two research groups analyzed preoperative stool samples in addition to generic hepatobiliary tissue obtained at surgery. Sample collection timing varied widely, with storage period prior to PCR experimentation not revealed in most reports on Reoviridae. CONCLUSION: Although a considerable number of PCR studies have sought to clarify a viral role in the pathogenesis of BA using human samples, the findings have been contradictory and have not succeeded in achieving an obvious differentiation between causative and accidental infection of the focused virus. Reproducible and convincing evidence for a causative Reoviridae infection has been lacking based on objective data from highly sensitive PCR experiments. Even though the possibility remains of viral disappearance at the timing of collection, to avoid further ambiguous interpretations of PCR results, rigorous and meticulous collection of large numbers of specimens at carefully planned timing, along with a strictly adjusted and finely tuned PCR system, is strongly recommended for obtaining more reliable and consistent results.
Assuntos
Atresia Biliar/virologia , Viroses/complicações , Humanos , Reação em Cadeia da Polimerase , Infecções por Reoviridae/complicações , Infecções por Reoviridae/diagnóstico , Viroses/diagnósticoRESUMO
BACKGROUND AND AIMS: Perinatal cytomegalovirus (CMV) infection is a possible cause or trigger of biliary atresia though clinical evidence is scant. We hypothesised that CMV IgM+ve biliary atresia is a separate clinical entity compared to CMV IgM-ve biliary atresia. METHODS: Prospective single-centre study. 210 infants with histologically confirmed biliary atresia were treated in our institution (Jan. 2004 to Dec. 2011); of these 20 (9.5%) were CMV IgM+ve at presentation. We compared these with 111 infants who were CMV IgM-ve (controls) for clinical features, biochemistry at presentation and outcome following Kasai portoenterostomy (KPE). A blinded comparison of age-matched liver histology was also performed. Data are quoted as median (interquartile range). A P value ≤ 0.05 was regarded as significant. RESULTS: Infants with CMV IgM+ve biliary atresia were older at Kasai portoenterostomy (or laparotomy) [70 (60-80) days vs. 56 (44-75)days; P = 0.003] and were more jaundiced [175 (147-224) vs. 140 (121-181) µmol/L; P = 0.002+ with higher AST*287 (157-403) vs. 180 (133-254) IU/L; P = 0.005] and aspartate aminotransferase-to-platelet ratio index [1.1 (0.79-3.0) vs. 0.63 (0.43-0.95)] levels. Liver histology: CMV IgM+ve biliary atresia was characterised by a greater degree of inflammation (P < 0.0001) and fibrosis (P = 0.02), whereas CMV IgM-ve isolated biliary atresia had a higher degree of lobular cholestasis (P = 0.001). This effect was independent of the effects of age at KPE. OUTCOME: CMV IgM+ve biliary atresia had a poorer outcome with a reduced clearance of jaundice (15% vs. 52.2%; P = 0.002), native liver survival (P < 0.0001) and increased mortality (P = 0.002). CONCLUSIONS: CMV IgM+ve biliary atresia is a distinct clinical and pathological entity with a diminished response to Kasai portoenterostomy.
Assuntos
Atresia Biliar/virologia , Infecções por Citomegalovirus/complicações , Fatores Etários , Aspartato Aminotransferases/sangue , Atresia Biliar/patologia , Atresia Biliar/cirurgia , Colestase/etiologia , Citomegalovirus/imunologia , Feminino , Humanos , Imunoglobulina M/análise , Lactente , Fígado/enzimologia , Fígado/patologia , Fígado/cirurgia , Masculino , Contagem de Plaquetas , Portoenterostomia Hepática , Prognóstico , Estudos ProspectivosRESUMO
BACKGROUND: Biliary atresia (BA), a cholangiopathy of unknown etiology is associated with intrahepatic mononuclear cell infiltrate. An abnormal reaction to viral exposure has been hypothesized in some cases. We aimed to investigate the nature of the CD4+ hepatic infiltrate in defined clinical variants of BA by quantification of inflammatory cell components. METHODS: Liver biopsies of infants obtained at Kasai portoenterostomy (KPE) were stained immunohistochemically using monoclonal antibodies to Tbet, GATA-3, FOXP3 and interleukin (IL) 17, identifying Th-1, Th-2, Tregs and Th-17 cells respectively. T cells were counted with the aid of a graticule. Data are reported as median (range) of cells per high-power-field (×400) and compared using nonparametric statistical tests with P≤0.05 regarded as significant. RESULTS: Liver biopsies from BA (n=37) and age-matched cholestatic controls (e.g. alpha-1-anti trypsin deficiency, Alagilles syndrome, n=12) were investigated. BA infants were divided into three groups: cytomegalovirus IgM +ve (CMV; n=9); BA splenic malformation (BASM; n=9) and isolated BA (IBA; n=19). All T-cell subsets were present in the portal tracts, with an overrepresentation of Th-1 (P<0.001) and Th-17 (P<0.03), but not Th-2 (P=0.94) or Tregs (P=0.15), compared to controls. Th-1 cells predominated in the CMV group; (18 [7-37] vs. 3 [0-14] [BASM] and vs. 5 [3-23] [IBA]; P<0.01 both), while no subgroup differences were seen for Th-17 cells. The degree of Th-1 cell infiltrate inversely correlated with platelet count (rS=-0.49; P<0.01). Th-17 cells were fewer (6 [2-11] vs. 11 [8-20]; P=0.02) in infants who cleared their jaundice (n=15, <20µmol/L) although this did not translate to improved native liver survival (P=0.17). CONCLUSIONS: Th-17 cells infiltrate the liver in BA and are associated with a worse surgical outcome; a Th-1 profile predominates in CMV-associated BA.
Assuntos
Atresia Biliar/imunologia , Fígado/imunologia , Portoenterostomia Hepática , Células Th17/metabolismo , Atresia Biliar/cirurgia , Atresia Biliar/virologia , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Humanos , Lactente , Recém-Nascido , Fígado/cirurgia , Fígado/virologia , Resultado do TratamentoRESUMO
BACKGROUND: The murine model of biliary atresia (BA) is used for examining the pathogenesis of BA. The aim of the study was description of the morphological features and illustrating the detailed development of fibrosis using the Biliary Atresia Research Consortium (BARC) system. METHODS: Neonatal mice were injected intraperitoneally with rhesus rotavirus (RRV) strain (N=17). Healthy mice were the control group (N=29). All mice were sacrificed at 7 or 14days after birth. Two pathologists examined the morphological features using the BARC system; CK19, αSMA and collagen type I were assessed by immunohistochemistry. RESULTS: In RRV mice, portal fibrous expansion with focal bile duct proliferation and strong portal cellular infiltrate was found in contrast to healthy mice. In RRV mice, CK19 bile duct staining was significantly less or absent (p<0.01), with stronger portal staining of collagen type I (p=0.02). Expansion of staining for αSMA was more in RRV mice (p<0.01), but αSMA portal staining was stronger in healthy mice (p=0.02). CONCLUSIONS: The morphological features observed in the murine model of BA correspond with the BA characteristics according to the BARC criteria. Fibrosis is an important feature of the model. Therefore, this murine model is useful for investigating the pathogenesis of BA.
Assuntos
Atresia Biliar/patologia , Cirrose Hepática/etiologia , Fígado/patologia , Animais , Atresia Biliar/virologia , Modelos Animais de Doenças , Progressão da Doença , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Rotavirus/complicações , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: To analyze the prevalence of acute asymptomatic group A and C rotavirus (RV-A and RV-C) infection in neonates with cholestasis. STUDY DESIGN: Participants were infants <180 days of age with cholestasis (serum direct or conjugated bilirubin >20% of total and ≥2 mg/dL) enrolled in the Childhood Liver Disease Research and Education Network during RV season (December-May). Forty infants with biliary atresia (BA), age 62 ± 29 days (range, 4.7-13 weeks) and 38 infants with cholestasis, age 67 ± 44 days (range, 3-15.8 weeks) were enrolled. RESULTS: At enrollment, RV-A IgM positivity rates did not differ between infants with BA (10%) vs those without (18%) (P = .349). RV-C IgM was positive in 0% of infants with BA vs 3% in those without BA (P = .49). RV-A IgG was lower in infants with BA: 51 ± 39 vs 56 ± 44 enzyme-linked immunoassay unit, P = .045 but this difference may lack biological relevance as maternal RV-A IgG titers were similar between groups. Infant RV-A IgM titers at 2-6 months follow-up increased markedly vs at presentation in both infants with BA (50 ± 30 vs 9 ± 9) and those without (43 ± 18 vs 16 ± 20 enzyme-linked immunoassay unit) (P < .0001), without differences between groups. CONCLUSIONS: RV-A infection in the first 6 months of life is common in infants with cholestasis of any cause. RV-A could have different pathogenetic effects by initiating different hepatic immune responses in infants with vs without BA or could lack pathogenetic significance.