| Biliary atresia(BA) is characterized as a destructive inflammatory obliterative cholangiopathy of infancy that affects varying lengths of both intrahepatic and extrahepatic bile ducts. It is most common in eastern Asian countries, with a reported frequency varying from1/5000to1/8000. Studies have shown that a geographic clustering of cases and seasonal variation, an association with advanced maternal age and increased parity, and a tendency for early fetal losses. A slight female predominance of1.25:1may be present. Biliary atresia is the most common cause of neonatal cholestasis in children and the prime indication for pediatric liver transplantation worldwide, accounting for about40%-50%of all liver transplants in children. Although surgical creation of the portoenterostomy (Kasai procedure) can improve some children with extrahepatic biliary obstruction, but70%to80%of patients with biliary atresia children eventually need a liver transplantation resulting from portal hypertension and liver cirrhosis. It is difficult for the pediatric surgeons to find out the way to improve long-term survival of children with biliary atresia, there is a necessity for research into the etiology and pathogenesis of biliary atresia in order to assess whether these aspects could be potential targets for therapeutic intervention.With the development of widely research, the application of genetics, immunology, and animal models to investigate biliary atresia have begun unraveling the contribution of infectious, immune, autoimmune, genetic, epigenetic, vascular and morphogenic processes in the etiology of biliary obstruction. Several pathogens have been identified in the patients with biliary atresia, such as human papillomavirus, respiratory syncytial virus, herpes virus, cytomegalovirus(CMV), reovirus type3and rotavirus. Of these, the rotavirus has received greater attention. Not only in the liver of biliary atresia detected rotavirus, newborn Balb/c-mice with rhesus rotavirus (RRV) by intraperitoneal injection also triggers tissue specific inflammation and features of biliary atresia and leads to a cholestasis and biliary obstruction resembling human biliary atresia. There are many similarities between animal models and human clinical and pathological features of children with B A, such as bile duct injury mediated by inflammatory cytokines, extrahepatic bile duct injury and the occurrence of morphological changes of the general time-dependent bile duct obstruction and so on. Therefore, it is likely that EHBE are a target of the rotavirus which directly cause the cholangiopathy in cases of biliary atresia.Rotaviruses (RV), family reoviridae, are non-enveloped virions with a genome of11double-stranded RNA segments. Six structural and six non-structural proteins are encoded by the segmented RNA genome. The early secretory diarrhea likely is induced by NSP4, which binds an integrin receptor and promotes calcium mobilization followed by a chloride secretory response. The hypothese is that NSP4is released from infected cells to interact with surface receptors of neighboring cells to trigger a specific signaling event that results in secretion. NSP4is the first described viral enterotoxin and encoded by RV gene10and the primary translation product of175amino acids with an apparent molecular weight of20kD glycoprotein, NSP4is a multi-functional glycoprotein that contributes to RV morphogenesis, replication and pathogenesis. RNA silencing (siRNA) studies of NSP4 expression reveal that in the absence of NSP4lead to an abnormal distribution of viral proteins in the viroplasm, little to no infectious viral particles in the cell, an accumulation of empty viral particles and an increase of viral transcripts, it is confirmed that NSP4is the main enterotoxin in the rotavirus-induced intestinal epithelia injury and the number of CD8+lymphocytes was significantly increased in peripheral blood.NSP4does not cause damage to the intestinal epithelium in mice with depletion of CD8+lymphocytes. CD8+lymphocytes is an important cytotoxic T lymphocytes (cytotoxic T lymphocytes, CTL) and the major cellular immune effector cells, it plays an important role in the anti-infective and anti-tumor immunity. Among the immune cells, CD8+lymphocyte activation plays an important role in the formation of biliary atresia in newborn mice induced by rotavirus. Studies have shown that after rotavirus infection, some of rotavirus antigen phagocytosed by macrophages then presentation to the CD4+lymphocytes, followed by activating CD8+lymphocytes, and secretion of perforin attack EHBE and lead to the destruction of the bile duct epithelial cells then antigen-presenting itself to the immune system, thus causing further damage. Newborn mice did not change the course of inflammatory injury and obstruction of neonatal bile ducts with depletion of CD4+cells. In contrast, depletion of CD8+lymphocytes surprisingly hampered duct injury, prevented luminal obstruction, and restored bile flow. It is concluded that primed neonatal CD8+lymphocytes can activate a proinflammatory program, target diseased and healthy duct epithelium, and drive the phenotypic expression of biliary atresia, thus leading to the destruction of extrahepatic bile ducts of neonatal mice.Cytotoxic T lymphocytes (CTL) epitopes are important for rational vaccine design, it is reported that only a small fraction of the peptides in a pathogen proteome are able to elicit a CTL response, the first step is the translocation of the peptides from the cytosol to the interior of the ER. The transport is facilitated by binding of the peptides to transporter associated with antigen presentation(TAP), followed by entering the ER, as well as binding of some of the peptides to MHC class I, after binding, the MHC class I-peptide complex is transported to the surface of the cell, where it may be recognized by CTL. Although there have been rotavirus VP6and VP7CTL epitopes reported, there is no rotavirus NSP4epitopes reported.It is hypothesed that primed neonatal CD8+lymphocytes by rotavirus NSP4can activate a proinflammatory program, target diseased and healthy duct epithelium, and leading to the destruction of extrahepatic bile ducts of neonatal mice.The aim of this study was to investigate the mechanism of rotavirus NSP4in the damage of extrahepatic biliary epithelia by utilizing NSP4and epitopes to activate the CD8+lymphocytes, followed by killing EHBE specifically and provide directions for reducing the incidence of biliary atresia by immune intervention and improving the prognosis of biliary atresia theoretically.PART ⅠThe role of integrins α2β1in rotavirus induced experimental cholangiocyte damageObjective:To investigate the expression and disposition of integrins on cultured biliary epithelia, and try to explore the role of integrins in rotavirus infection in vitro.Methods:Extrahepatic biliary epithelia were cultured for3days. The primary cultured epithelia were fixed and immunofluorescent (IF) assay was performed to detect integrin subunits of α2, α4and β1. The conjugation of rotavirus and biliary epithelia was observed under transmission electron microscope (TEM). After different integrin antibodies were incubated on cultured cells for1h (PBS incubated on cultured cells served as the negative control), RRV was inoculated. Cytopathic effect (CPE) of different groups was observed under microscope and analyzed by SNK-test.Results:The subunits of α2, α4and β1were detected at the surface of extrahepatic biliary epithelia. The conjugation of rotavirus and biliary epithelia was observed under TEM. Significantly decreased CPE of cultured cells at6hpi was found in the antibody incubated groups, p<0.01compared to that of the negative control group; CPE in α2β1blocking group was significantly decreased, p<0.01compared to that in the α4β1blocking group. At24hpi, significantly decreased CPE was also noted in the α2β1blocking group, p<0.01compared to that in the negative control group.Conclusions:The integrin subunits of α2, α4and β1were expressed and located on the surface of primary cultured biliary epithelia. The integrin α2β1and α4β1can mediate the RV infection and injury of biliary epithelia. Blocking α2β1may have more contribution in preventing biliary epithelia from injury caused by rotavirus infection than blocking α4β1. The integrins may be new targets for therapy of BA. PART ⅡThe role of rotavirus NSP4and integrin α2β1in the damage of extrahepatic biliary epitheliaObjective:To investigate the role of rotavirus NSP4(non structural protein4, NSP4) and integrin a2(31in the damage of extrahepatic biliary epithelia.Methods:Extrahepatic biliary epithelia were cultured for3days. The primary cultured epithelia were fixed and immunofluorescent (IF) assay was performed to detect integrin subunits of a2, β1and rotavirus nonstructural protein4(NSP4). Small interfering RNA targeting integrin a2was transfected before rotavirus infection in vitro. Real-time RT-PCR was performed for measurements of integrin a2and NSP4mRNAs, the value of2-△△CTwas expressed as mean±SD from3independent experiments and analyzed by SNK-test.Results:The subunits of integrin a2and (β were detected at the surface of extrahepatic biliary epithelia. NSP4colocalized with integrin α2on the surface of rotavirus-infected extrahepatic biliary epithelia, indicating that NSP4released from rotavirus-infected cells binds the integrin a2on adjacent uninfected cells. Silencing of integrin α2decreased the messenger RNA (mRNA) levels of rotavirus NSP4.Conclusions:The integrin subunits of α2, β1were expressed and located on the surface of primary cultured extrahepatic biliary epithelia. The integrin α2β1was receptor for the rotavirus NSP4and mediate the injury of extrahepatic biliary epithelia. PART ⅢThe role of rotavirus NSP4in determining the injury of biliary atresia in murine modelObjective:The goal of this part was to investigate the mechanism of rotavirus nonstructural protein4(NSP4) that governs the induction of murine BA.Methods:Within the first24hours of life, the newborn mice were immunized through intraperitoneal route with NSP4.The methods of intracellular cytokine staining and ELISPOT were used to detect IFN-γ production by liver CD8+cells and lymphocytes at7days and14days post-immunized. Cytotoxicity of liver CD8+cells against cultured extrahepatic biliary epithelia was measured by the CytoTox96nonradioactive cytotoxicity assay. In peripheral blood serum, the method of ELISA was used to detect the expression of IFN-γ. The production of IFN-γ was investigated by liver lymphocytes and serum of peripheral blood after depleting CD8+T cells. The newborn mice were infected through intraperitoneal route with rotavirus NSP4followed by depleting CD8+T cells, then the pups were sacrificed on day7after inoculation and extrahepatic bile ducts of specimens were stained with H&E and used for histopathologic studies.Results:Expression of intracellular IFN-γ production by liver CD8+cells at7days and14days postinfection were all significantly up-regulated in the group of NSP4(P<0.05). The expression of IFN-γ was greatly up-regulated in the group of NSP4by liver lymphocytes with ELISPOT (P<0.05). Highly activated liver CD8+cells were cytotoxic to cultured extrahepatic biliary epithelia was markedly up-regulated in the group of NSP4(P<0.05), and the cytoxicity of liver CD8+cells directed against infected cultured extrahepatic biliary epithelia was significantly greater than that directed against uninfected cultured extrahepatic biliary epithelia(P<0.05). In peripheral blood serum, the expression of IFN-γ was greatly up-regulated in the group of NSP4(P<0.05). The expression of IFN-γ was significantly diminished by liver lymphocytes and serum of peripheral blood with depletion of CD8+cells (P<0.05).In vivo experiments indicated after depleting CD8+T cells, mouse pups had lower incidence of BA than control group.Conclusions:Rotavirus NSP4-primed neonatal CD8+cells play an important role in the damage of extrahepatic bile ducts. PART IVRoles of rotavirus NSP4epitopes in governing the immune system of biliary atresia in murine modelObjective:To investigate and identify the rotavirus nonstructural protein4(NSP4) epitopes that governs the immune system.Methods:Within the first24hours of life, the newborn mice were immunized through intraperitoneal route with NSP4epitopes by solid phase peptide synthesis with bioinformaticians.The methods of intracellular cytokine staining and ELISPOT were used to detect IFN-γ production by liver CD8+cells and lymphocytes at7days and14days post-immunized. Cytotoxicity of liver CD8+cells against cultured extrahepatic biliary epithelia was measured by the CytoTox96nonradioactive cytotoxicity assay. In peripheral blood serum, the method of ELIS A was used to detect the expression of IFN-γ.Results:Expression of intracellular IFN-γ production by liver CD8+cells at7days and14days postinfection were all significantly up-regulated in the groups of NSP4157-170and NSP4144-152than NSP424-32and NSP493-110(P<0.05). The production of IFN-γ was greatly increased in the groups of NSP4157-170and NSP4144-152than NSP424-32and NSP493-110by liver lymphocytes with ELISPOT (P<0.05). Highly activated liver CD8+cells were cytotoxic to cultured extrahepatic biliary epithelia was markedly up-regulated in the groups of NSP4157-170and NSP4144-152than NSP424-32and NSP493-110(P<0.05), and the cytoxicity of liver CD8+cells directed against infected cultured extrahepatic biliary epithelia was significantly greater than that directed against uninfected cultured extrahepatic biliary epithelia(P<0.05). In peripheral blood serum, the expression of IFN-γ was greatly up-regulated in the groups of NSP4157-170and NSP4144-152than NSP424-32and NSP493-110(P<0.05).Conclusions:NSP4157-170and NSP4144-152may be the CTL epitopes of the rotavirus NSP4. |
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