Unique Cholangiocyte-Targeted IgM Autoantibodies Correlate With Poor Outcome in Biliary Atresia.

BACKGROUND AND AIMS: The etiology of biliary atresia (BA) is not known and is likely multifactorial, including a genetic predisposition, a viral or environmental trigger, an aberrant autoimmune response targeting cholangiocytes, and unique susceptibilities of the neonatal bile ducts to injury. Damaged cholangiocytes may express neo self-antigens and elicit autoreactive T-cell-mediated inflammation and B-cell production of autoantibodies. The aim of this study was to discover autoantibodies in BA that correlated with outcomes. APPROACH AND RESULTS: An autoantigen microarray encompassing approximately 9,500 autoantigens was used to screen for serum immunoglobulin M (IgM) and immunoglobulin G (IgG) autoantibodies in patients with BA or other liver disease controls. Validation of candidate autoantibodies by enzyme-linked immunosorbent assay on a second cohort of subjects (6-12 months following Kasai portoenterostomy) and correlations of autoantibodies with outcomes were performed (serum bilirubin levels and need for liver transplant in first 2 years of life). Mean anti-chitinase 3-like 1 (CHI3L1), anti-delta-like ligand (DLL-4), and antisurfactant protein D (SFTPD) IgM autoantibodies in BA were significantly higher compared with controls, and IgM autoantibody levels positively correlated with worse outcomes. Immunofluorescence revealed cholangiocyte-predominant expression of CHI3L1, DLL-4, and SFTPD. The humoral autoantibody response was associated with C3d complement activation and T-cell autoimmunity, based on detection of cholangiocyte-predominant C3d co-staining and peripheral blood autoreactive T cells specific to CHI3L1, DLL-4 and SFTPD, respectively. CONCLUSIONS: BA is associated with cholangiocyte-predominant IgM autoantibodies in the first year after Kasai portoenterostomy. Anti-CHI3L1, anti-DLL-4, and anti-SFTPD IgM autoantibody correlations with worse outcomes and the detection of C3d on cholangioctyes and antigen-specific autoreactive T cells suggest that autoimmunity plays a role in the ongoing bile duct injury and progression of disease.

Single cell RNA sequencing reveals regional heterogeneity of hepatobiliary innate lymphoid cells in a tissue-enriched fashion.

IL-33 promotes type 2 immunity, epithelial repair, and tissue fibrosis by activating group 2 innate lymphoid cells (ILC2). ILC2 lack all known surface markers of mature T, B, NK, and myeloid cell lineages (Linneg), express the IL-33 receptor ST2, and release type 2 cytokines which contribute to cholangiocyte proliferation and activation of hepatic stellate cells. This pathway results in massive proliferation of the extrahepatic bile duct (EHBD) but also exacerbates liver fibrosis, suggesting that there may be tissue-specific subpopulations of IL-33-induced ILC. To determine the tissue-specific subsets of ILC in the hepatobiliary system, we analyzed CD45+Linneg mononuclear cells from IL-33 treated adult Balb/c mouse liver or EHBD by single cell RNA sequencing. Principal component analysis identified 6 major CD45+Linneg cell classes, two of which were restricted to the EHBD. One of these classes, biliary immature myeloid (BIM) cells, was predicted to interact with ILC2 by a network of shared receptor-ligand pairs. BIM highly expressed Gp49 and ST2 receptors on the cell surface while lacking surface expression of markers for mature myeloid cells. In conclusion, single cell RNA sequencing identified IL-33 responsive cell groups regionally confined to the liver or extrahepatic bile duct, including a novel population of CD45+Linneg Gp49-expressing mononuclear cells.

Clinical Implications of Cytotoxic T Lymphocyte Antigen-4 Expression on Tumor Cells and Tumor-Infiltrating Lymphocytes in Extrahepatic Bile Duct Cancer Patients Undergoing Surgery Plus Adjuvant Chemoradiotherapy.

BACKGROUND: There currently is only limited knowledge on the role of tumor-specific immunity in cholangiocarcinoma. OBJECTIVE: This study evaluated the clinical implications of cytotoxic T lymphocyte antigen-4 (CTLA-4) expression levels and CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) in extrahepatic bile duct (EHBD) cancer. PATIENTS AND METHODS: Immunohistochemistry of CTLA-4, CD4, and CD8 was performed for 77 EHBD cancer patients undergoing surgery plus adjuvant chemoradiotherapy. CTLA-4 expression on tumor cells and TILs were assessed by using H-scores and the proportion of CTLA-4+ lymphocytes, respectively. RESULTS: With optimal cutoff values determined by a maximal chi-square method with overall survival (OS) data, patients with CTLA-4 H-score >70 and a proportion of CTLA-4+ TILs >0.15 showed higher mean density of CD8+ and CD4+ TILs, respectively (P = 0.025 for CD8+ and P = 0.055 for CD4+ TILs). The high CTLA-4 H-score level was associated with prolonged OS and disease-free interval (DFI) (P = 0.025 and 0.004, respectively). With differential levels of CTLA-4 H-score according to hilar and non-hilar locations (high rate 32 vs. 68%, respectively; P = 0.013), an exploratory subgroup analysis demonstrated that the associations between the CTLA-4 expression and OS and DFI were confined to hilar tumors (P = 0.003 and <0.001, respectively), but not to non-hilar ones (P = 0.613 and 0.888, respectively). CONCLUSIONS: This study demonstrates a potential prognostic relevance of CTLA-4 expression in EHBD cancer. We suggest a differential survival impact of the CTLA-4 expression level according to different tumor locations.

Prognostic Impact of CD163+ Macrophages in Tumor Stroma and CD8+ T-Cells in Cancer Cell Nests in Invasive Extrahepatic Bile Duct Cancer.

AIM: The aim of this study was to examine the clinicopathological influence of tumor-infiltrating cluster of differentiation (CD) 163+ macrophages and CD8+ T-cells, and to clarify the prognostic effects of these cells in patients with invasive extrahepatic bile duct cancer (EHBC). MATERIALS AND METHODS: The numbers of CD8+ T-cells in cancer cell nests and CD163+ macrophages in tumor stroma were evaluated using immunohistochemistry in 101 resected EHBC specimens. Correlations with clinicopathological variables and overall survival were analyzed. RESULTS: Perihilar EHBC and perineural invasion were significantly associated with a low number of tumor-infiltrating CD8+ T-cells. Poorly- differentiated histology and nodal metastasis were significantly associated with a high number of tumor-infiltrating CD163+ macrophages. A combination of high number of CD8+ T-cells and low number of CD163+ macrophages was independently related to better overall survival in the whole patient cohort (hazard ratio=0.127, p<0.001) and in patients treated with adjuvant chemotherapy (hazard ratio=0.139, p=0.021). CONCLUSION: Infiltrating CD163+ macrophages in tumor stroma and CD8+ T-cells in cancer cell nests have a prognostic impact in patients with EHBC following resection and also after adjuvant chemotherapy.

Implicación pronóstica de las manifestaciones clínicas extrahepáticas, autoinmunidad y capilaroscopia ungueal microscópica en pacientes con cirrosis biliar primaria / Prognostic implications of extra-hepatic clinical manifestations, autoimmunity and microscopic nail capillaroscopy in patients with primary biliary cirrhosis

Introducción y objetivos: La cirrosis biliar primaria (CBP) se asocia a algunas enfermedades autoinmunes sistémicas (EAS), en particular a la esclerosis sistémica (ES. Determinar la prevalencia de EAS en una cohorte de pacientes con CBP, específicamente la ES y sus diferentes subtipos clínicos, y establecer el perfil clínico-biológico propio de estos pacientes. Métodos: Estudio observacional de 62 pacientes con CBP, con un protocolo que incluía una anamnesis y exploración física dirigidas a detectar una EAS, la realización de una capilaroscopia ungueal microscópica y un amplio estudio de autoinmunidad, incluido el perfil de anticuerpos específicos de ES. Se realizó un análisis comparativo entre el grupo de pacientes con CBP aislada y los pacientes con CBP y una EAS asociada. Resultados: Se asoció una EAS en 22 pacientes (35,4%), y la ES fue la entidad más frecuente (21%), del subtipo cutáneo limitado (11%). Cinco pacientes (8%) sin EAS previa cumplían criterios de preesclerodermia, según los criterios de LeRoy y Medsger. Los anticuerpos anticentrómero (54,5 vs. 5%, p < 0,001) fueron el único parámetro inmunológico identificado con mayor frecuencia en pacientes con EAS. El patrón capilar sugestivo de ES se visualizó en 11 pacientes (20,4%). No se identificaron factores asociados a mayor morbimortalidad en ningún grupo. Conclusiones: Existe un subgrupo de pacientes con CBP con características clínico-biológicas que sugieren la asociación con una EAS, con elevada probabilidad, y que recomiendan el estudio protocolizado de estos pacientes con CBP para detectar de forma precoz EAS (AU)

Background and objectives: Primary biliary cirrhosis (PBC) is associated to any systemic autoimmune disease (SAD), in particular systemic sclerosis (SSc). To investigate the prevalence of SAD in a cohort of patients with PBC, specifically the prevalence of SSc and its clinical subtypes, and determining the clinical and biological profile of patients with associated PBC and SSc. Methods: Observational study of 62 patients with PBC following a protocol that included an anamnesis and physical examination to detect the presence of SAD as well as a nailfold capillaroscopy and an immunological study with specific SSc autoantibodies. A comparative analysis was conducted between patients with isolated PBC and patients with PBC and an associated SAD. Results: SAD was associated to PBC in 22 patients (35,4%), and SSc was the most frequent illness, identified in 13 cases (21%). Five patients (8%) without previous diagnosis of SAD fulfilled pre-scleroderma criteria, according to LeRoy and Medsger criteria. The presence of anticentromere antibodies (54,5% vs. 5%,P < .001) was the unique immunological determination identified more frequently in patients with PBC-SAD. The SSc suggestive capillary pattern was visualized in 11 patients (20,4%), mainly the slow pattern. No factors associated with greater morbi-mortality were identified in the PBC-SAD group. Conclusions: It does exist a subgroup of patients with PBC and clinical-biological features suggestive of an SAD, which advise a protocolized study to detect early the association to an SAD (AU)

Biliary repair and carcinogenesis are mediated by IL-33-dependent cholangiocyte proliferation.

Injury to the biliary epithelium triggers inflammation and fibrosis, which can result in severe liver diseases and may progress to malignancy. Development of a type 1 immune response has been linked to biliary injury pathogenesis; however, a subset of patients with biliary atresia, the most common childhood cholangiopathy, exhibit increased levels of Th2-promoting cytokines. The relationship among different inflammatory drivers, epithelial repair, and carcinogenesis remains unclear. Here, we determined that the Th2-activating cytokine IL-33 is elevated in biliary atresia patient serum and in the livers and bile ducts of mice with experimental biliary atresia. Administration of IL-33 to WT mice markedly increased cholangiocyte proliferation and promoted sustained cell growth, resulting in dramatic and rapid enlargement of extrahepatic bile ducts. The IL-33-dependent proliferative response was mediated by an increase in the number of type 2 innate lymphoid cells (ILC2s), which released high levels of IL-13 that in turn promoted cholangiocyte hyperplasia. Induction of the IL-33/ILC2/IL-13 circuit in a murine biliary injury model promoted epithelial repair; however, induction of this circuit in mice with constitutive activation of AKT and YAP in bile ducts induced cholangiocarcinoma with liver metastases. These findings reveal that IL-33 mediates epithelial proliferation and suggest that activation of IL-33/ILC2/IL-13 may improve biliary repair and disruption of the circuit may block progression of carcinogenesis.

CD8+ T lymphocyte response against extrahepatic biliary epithelium is activated by epitopes within NSP4 in experimental biliary atresia.

Interferon (IFN)-γ-driven and CD8+ T cell-dependent inflammatory injury to extrahepatic biliary epithelium (EHBE) is likely to be involved in the development of biliary atresia (BA). We previously showed that viral protein NSP4 is the pathogenic immunogen that causes biliary injury in BA. In this study, NSP4 or four synthetic NSP4 (NSP4(157-170), NSP4(144-152), NSP4(93-110), NSP4(24-32)) identified by computer analysis as candidate CD8+ T cell epitopes were injected into neonatal mice. The pathogenic NSP4 epitopes were confirmed by studying extrahepatic bile duct injury, IFN-γ release and CD8+ T cell response against EHBE. The results revealed, at 7 days postinjection, inoculation of glutathione S-transferase (GST)-NSP4 caused EHBE injury and BA in neonatal mice. At 7 or 14 days postinject, inoculation of GST-NSP4, NSP4(144-152), or NSP4(157-170) increased IFN-γ release by CD8+ T cells, elevated the population of hepatic memory CD8+ T cells, and augmented cytotoxicity of CD8+ T cells to rhesus rotavirus (RRV)-infected or naive EHBE cells. Furthermore, depletion of CD8+ T cells in mice abrogated the elevation of GST-NSP4-induced serum IFN-γ. Lastly, parenteral immunization of mouse dams with GST-NSP4, NSP4(144-152), or NSP4(157-170) decreased the incidence of RRV-induced BA in their offspring. Overall, this study reports the CD8+ T cell response against EHBE is activated by epitopes within rotavirus NSP4 in experimental BA. Neonatal passive immunization by maternal vaccination against NSP4(144-152) or NSP4(157-170) is effective in protecting neonates from developing RRV-related BA.

CD24 expression predicts distant metastasis in extrahepatic bile duct cancer.

AIM: To evaluate the prognostic significance of CD24 expression in patients undergoing adjuvant chemoradiotherapy for extrahepatic bile duct (EHBD) cancer. METHODS: Eighty-four patients with EHBD cancer who underwent curative resection followed by adjuvant chemoradiotherapy were enrolled in this study. Postoperative radiotherapy was delivered to the tumor bed and regional lymph nodes up to a median of 40 Gy (range: 40-56 Gy). All patients also received fluoropyrimidine chemotherapy for radiosensitization during radiotherapy. CD24 expression was assessed with immunohistochemical staining on tissue microarray. Clinicopathologic factors as well as CD24 expression were evaluated in multivariate analysis for clinical outcomes including loco-regional recurrence, distant metastasis-free and overall survival. RESULTS: CD24 was expressed in 36 patients (42.9%). CD24 expression was associated with distant metastasis, but not with loco-regional recurrence nor with overall survival. The 5-year distant metastasis-free survival rates were 55.1% and 29.0% in patients with negative and positive expression, respectively (P = 0.0100). On multivariate analysis incorporating N stage, histologic differentiation and CD24 expression, N stage was the only significant factor predicting distant metastasis-free survival (P = 0.0089), while CD24 expression had borderline significance (P = 0.0733). In subgroup analysis, CD24 expression was significantly associated with 5-year distant metastasis-free survival in node-positive patients (38.4% with negative expression vs 0% with positive expression, P = 0.0110), but not in node-negative patients (62.0% with negative expression vs 64.0% with positive expression, P = 0.8599). CONCLUSION: CD24 expression was a significant predictor of distant metastasis for patients undergoing curative resection followed by adjuvant chemoradiotherapy especially for node-positive EHBD cancer.

Pathologic significance of immunoglobulin G4-positive plasma cells in extrahepatic cholangiocarcinoma.

Immunoglobulin G4-related sclerosing cholangitis is histologically characterized by the infiltration of immunoglobulin G4-positive plasma cells and sclerosing change. Moreover, several cases of carcinoma accompanied by immunoglobulin G4-positive cells in tissue and increased serum immunoglobulin G4 levels have been reported, but the association between cancer-associated immunity and an immunoglobulin G4 reaction is still unclear. In this study, we examined the infiltration of immunoglobulin G4-positive cells in extrahepatic cholangiocarcinoma and the pathologic significance of the immunoglobulin G4 reaction found in cancer tissues in terms of the evasion of immune surveillance by regulatory T cells. Immunohistochemistry for immunoglobulin G4, forkhead box P3, CD4, and CD8 was performed using 68 surgical specimens from patients with extrahepatic cholangiocarcinoma, and positive cells were investigated, particularly within and around cancerous tissues. Consequently, although immunoglobulin G4+ cells were few (average, <10 cells/high-power field) in most cases, 10 or more and 50 or more cells were found in 37% and 6% of cases, respectively. Immunoglobulin G4+ cells were predominantly found in the invasive front of carcinoma tissue. In the cases with 10 or more immunoglobulin G4+ cells, forkhead box P3+ regulatory T cells were also distinguishable, and a positive correlation was found between the forkhead box P3+/CD4+ ratio and immunoglobulin G4+ cell count, but few CD8+ cells invaded cancer cells (<10 cells). In conclusion, extrahepatic cholangiocarcinomas are often accompanied by the significant infiltration of immunoglobulin G4+ cells, and the immunoglobulin G4 reaction showed a positive and negative correlation with forkhead box P3+ and CD8+ cells, respectively, suggesting the evasion of immune surveillance associated with CD8+ cytotoxic T cells via the regulatory function of forkhead box P3+ regulatory T cells.

Epidemiological survey of biomarkers of hepatitis virus in patients with extrahepatic cholangiocarcinomas.

AIM: Hepatitis virus B and C (HBV and HCV) are suggested to be risk factors for intrahepatic cholangiocarcinoma (ICC), but whether they are risk factors for extrahepatic cholangiocarcinoma (ECC) is disputed. To test the biomarkers in patients with ECC and further elucidate the relationship of HBV or HCV infection with ECC risk, we conducted a retrospective survey on hepatitis virus markers in patients with ECC. METHODS: A hospital-based case-control study was conducted to review prior infection with hepatitis virus and the seroprevalence of hepatitis virus markers in the patients with ECC or with benign biliary disease (BBD). HBV X antigen (HBxAg) was detected in the tissues by immunohistochemical staining. RESULTS: A total of 305 patients with ECC and 480 with BBD were enrolled in this study. Compared with BBD patients, ECC patients had a higher prevalence of prior infection with HBV (6.2 vs 2.3%) and chronic HBV infection (9 vs 1.9%). The overall seropositive rate for HBV markers in the two groups was 22.6 versus 6% (P < 0.01) and for HBxAg detection it was 75 versus 26% (P < 0.001). The seroprevalence of anti-HCV was 4.3% in the EEC patients and 5.6% in BBD patients with no significant difference between them. CONCLUSION: The high prevalence of HBV biomarkers in ECC strongly supports the notion that HBV infection may be a risk factor for ECC. The high frequency of HBxAg expression suggests its important role in the pathogenesis of bile duct neoplasm.

Biliary atresia.

Biliary atresia (BA) is a condition unique to infancy. It results from inflammatory destruction of the intrahepatic and extrahepatic bile ducts. It is the most frequent surgically correctable liver disorder in infancy and the most frequent indication for liver transplantation in paediatric age. Clinical presentation is in the first few weeks of life with conjugated hyperbilirubinaemia (dark urine and pale stools); other manifestations of liver disease, such as failure to thrive, splenomegaly and ascites, appear only later, when surgery is unlikely to be successful. Hence, all infants with conjugated hyperbilirubinaemia must be urgently referred to specialised centres for appropriate treatment. Success of surgery depends on the age at which it is performed. With corrective surgery, followed, when necessary, by liver transplantation, the overall survival rate is approximately 90%. The cause of BA is unknown, but there is evidence for the involvement of infectious, genetic and immunologic mechanisms, which will be discussed in this review.

Cholangiocytes as immune modulators in rotavirus-induced murine biliary atresia.

BACKGROUND/AIMS: Biliary atresia (BA) is a progressive disease characterized by bile duct inflammation and fibrosis. The aetiology is unknown and may be due to a virus-induced, autoimmune-mediated injury of cholangiocytes. Cholangiocytes are not only targets of injury but may also modulate hepatic inflammation. The aim of this study was to determine the immune profile of murine cholangiocytes and the ability to function as antigen-presenting cells (APCs) in culture with Rhesus rotavirus (RRV), poly I:C (viral mimic) or interferon-gamma/tumour necrosis factor-alpha. METHODS/RESULTS: Both the cholangiocyte cell line (long-term culture) and fresh, ex vivo cholangiocytes expressed APC surface markers major histocompatibility complex (MHC)-class I and II and CD40, while only the cultured cell line expressed costimulatory molecules B7-1 and B7-2. Despite APC expression, cultured cholangiocytes were unable to function as competent APCs in T-cell proliferation assays. Furthermore, both cultured and ex vivo cholangiocytes expressed RNA transcripts for many pro-inflammatory cytokines and chemokines. CONCLUSIONS: Although cholangiocytes contain APC molecules, they are incompetent at antigen presentation and cannot elicit effective T-cell activation. Upregulation of MHC-class I and II found in BA mice may serve to prime the cholangiocyte as a target for immune-mediated injury. Cholangiocytes produced many pro-inflammatory cytokines and chemokines in the setting of RRV infection and T-helper type 1 cytokine milieu, suggesting a role of cholangiocytes as immune modulators promoting the ongoing inflammation that exists in RRV-induced BA.

Oligoclonal expansions of CD4+ and CD8+ T-cells in the target organ of patients with biliary atresia.

BACKGROUND & AIMS: Biliary atresia is an inflammatory, fibrosclerosing neonatal cholangiopathy, characterized by a periductal infiltrate composed of CD4(+) and CD8(+) T cells. The pathogenesis of this disease has been proposed to involve a virus-induced, subsequent autoreactive T cell-mediated bile duct injury. Antigen-specific T-cell immunity involves clonal expansion of T cells expressing similar T-cell receptor (TCR) variable regions of the beta-chain (Vbeta). We hypothesized that the T cells in biliary atresia tissue expressed related TCRs, suggesting that the expansion was in direct response to antigenic stimulation. METHODS: The TCR Vbeta repertoire of T cells from the liver, extrahepatic bile duct remnants, and peripheral blood of biliary atresia and other cholestatic disease controls were characterized by fluorescent-activated cell sorter analysis, and TCR junctional region nucleotide sequencing was performed on expanded TCR Vbeta regions to confirm oligoclonality. RESULTS: FACS analysis revealed Vbeta subset expansions of CD4(+) and CD8(+) T cells from the liver or bile duct remnant in all patients with biliary atresia and only 1 control. The CD4(+) TCR expansions were limited to Vbeta3, -5, -9, and -12 T-cell subsets and the CD8(+) TCR Vbeta expansions were predominantly Vbeta20. Each Vbeta subset expansion was composed of oligoclonal populations of T cells. CONCLUSIONS: Biliary atresia is associated with oligoclonal expansions of CD4(+) and CD8(+) T cells within liver and extrahepatic bile duct remnant tissues, indicating the presence of activated T cells reacting to specific antigenic stimulation. Future studies entail identifying the specific antigen(s) responsible for T-cell activation and bile duct injury.

Immune-mediated liver injury: prognostic value of CD4+, CD8+, and CD68+ in infants with extrahepatic biliary atresia.

BACKGROUND: Hepatic fibrosis and cirrhosis develop progressively in extrahepatic biliary atresia (EHBA) despite timely surgical intervention. PURPOSE: The aim of the study was to define CD4+ helper T lymphocytes, cytotoxic CD8+ T lymphocytes, and CD68+ (macrophages) infiltration of portal tracts and lobules and hepatic fibrosis as possible predictive measures of outcome of infants having EHBA. METHODS: The outcome of 32 infants with EHBA was correlated to their percutaneous biopsy and postportoenterostomy core liver tissue infiltration by CD4+, CD68+, and CD8+ cells and to the degree of detected fibrosis. RESULTS: Portoenterostomy cores were heavily infiltrated by CD4+, CD8+, and CD68+, compared with the preoperative liver biopsy (P = .008, .004, and .017, respectively). Infants having favorable outcome had more macrophage infiltration in portoenterostomy core compared with those having an unfavorable outcome (25.66 +/- 29.77 per HPF compared with 11.62 +/- 4.58, P = .000). Mean CD4+/CD8+ ratio was 1.54 +/- 1.37 in those who died within 18 months postoperatively and 0.733 +/- 0.48 in others (P = .021). CONCLUSION: Immune-mediated destruction of portal tracts is an integral part of pathogenesis of EHBA.

Biliary atresia is associated with CD4+ Th1 cell-mediated portal tract inflammation.

A proposed mechanism in the pathogenesis of biliary atresia involves an initial virus-induced, progressive T cell-mediated inflammatory obliteration of bile ducts. The aim of this study was to characterize the inflammatory environment present within the liver of infants with biliary atresia to gain insight into the role of a primary immune-mediated process versus a nonspecific secondary response to biliary obstruction. Frozen liver tissue obtained from patients with biliary atresia, neonatal giant cell hepatitis, total parenteral nutrition (TPN)-related cholestasis, choledochal cysts, and normal control subjects was used for fluorescent immunohistochemistry studies of cellular infiltrates, cytokine mRNA expression, and in situ hybridization for localization of cytokine-producing cells. Immunohistochemistry revealed increases in CD8(+) and CD4(+) T cells and Kupffer cells (CD68(+)) in the portal tracts of biliary atresia. Reverse transcription-PCR analysis of biliary atresia tissue showed a Th1-type cytokine profile with expression of IL-2, interferon-gamma, tumor necrosis factor-alpha, and IL-12. This profile was not seen in normal, neonatal hepatitis or choledochal cyst livers but was present in TPN-related cholestasis. In situ hybridization revealed that the Th1 cytokine-producing cells were located in the portal tracts in biliary atresia and in the parenchyma of TPN-related cholestasis. A distinctive portal tract inflammatory environment is present in biliary atresia, involving CD4(+) Th1 cell-mediated immunity. The absence of similar inflammation in other pediatric cholestatic conditions suggests that the portal tract inflammation in biliary atresia is not a secondary response to cholestasis but rather indicates a specific immune response involved in the pathogenesis of biliary atresia.

Cytomegalovirus infection and proinflammatory cytokine activation modulate the surface immune determinant expression and immunogenicity of cultured murine extrahepatic bile duct epithelial cells.

Murine extrahepatic bile duct epithelial cells (MEBEC) were isolated from extrahepatic bile ducts of BALB/c mice and established in primary culture. The epithelial origin was confirmed by positive cytokeratin 19 staining for these cells and the presence of microvilli and tight junctions under electron microscopy. By immunofluorescent staining with monoclonal antibodies and flow-cytometric analysis, MEBEC in culture constitutively express low levels of intercellular adhesion molecule (ICAM)-1, class I and class II major histocompatibility (MHC) antigens. The expression of ICAM-1 was significantly increased by interferon gamma (INF-gamma) or tumour necrosis factor alpha (TNF-alpha) stimulation. Class I and class II antigen expression were significantly enhanced by INF-gamma and in vitro murine cytomegalovirus (MCMV) infection. MEBEC infected with MCMV revealed a progressive cytopathic effect. MEBEC activated by INF-gamma or infected by MCMV induced a low but significant proliferation of allogeneic T cells and displayed a significant decrease in the absorbance at O.D. 550 nm in a microtitre tetrazolium assay after these treated cells were co-cultured with allogeneic T cells. These results suggest that following the up-regulation of surface MHC antigen and adhesion molecule expression with cytokines or MCMV, the MEBEC can function as antigen-presenting cells and initiate T-cell proliferation, which in turn trigger the recognition of MEBEC by effector T-cell-mediated cytotoxic responses. These findings may be implicated in the pathogenesis of virally induced, immune-mediated extrahepatic bile duct damage disorders.

Progress in developing animal models for biliary atresia.

Animal models for extrahepatic biliary atresia (EHBA) have failed to simulate the course of the disease. Until now only a few aspects of the entity could be investigated and no model was helpful in discovering the etiology of EHBA. Following the suspicion of a viral and hepatotropic infection, investigations in an infectious mouse model were continued. The results of previous and topical studies are summarized here. Infection of newborn Balb/c-mice with rhesus rotavirus (RRV) leads to cholestasis in 85% of the animals followed by a lethality of 90%. Preparation and histomorphological investigation of liver and ligamentum duodenale reveal EHBA of varying extent. Clinical course and morphological findings in mice are very similar to EHBA in newborn children and the results are presented in a chronological table. Hepatobiliary morbidity and lethality after RRV infection is higher in Balb/c-mice than in other mouse strains. This observation supports the suspicion that immunocompetence might be a determining factor in the etiology of EHBA. Initial therapeutic trials were made using this model by treating infected newborn mice with interferon-alpha (IFN). The prophylactic application of IFN protects the infected mice from cholestatic symptoms and appears to induce partial immunity. Their descendants are protected against the hepatotropic effect of RRV infection. Infected animals presenting with clinical signs of cholestasis can be treated successfully by IFN-therapy for one week. In the presented animal model. EHBA can be better induced and simulated than by any other method. As a first trial, a non-surgical and more etiologically orientated therapeutic method is tested in this model.

Close anatomical relationships between nerve fibers and MHC class II-expressing dendritic cells in the rat liver and extrahepatic bile duct.

The anatomical relationships between immunocytochemically identified nerve fibers and MHC class II-expressing antigen presenting dendritic cells were investigated in the rat hepatobiliary system using immunocytochemistry, confocal laser scanning, and electron microscopy. Close proximity of nerve fiber varicosities immunostained for PGP 9.5 and MHC class II-expressing dendritic cells was frequently observed in the wall of extrahepatic bile ducts, in Glisson's area, around central and hepatic veins, and in the liver capsule. Contacts between nerve fibers staining for substance P, calcitonin gene-related peptide, calretinin, and vasoactive intestinal polypeptide and dendritic cells were more often observed around extrahepatic bile ducts than in Glisson's area. Nerve fibers immunostaining for tyrosine hydroxylase and neuropeptide Y were numerous both in the wall of extrahepatic bile ducts and in Glisson's area and frequently contacted dendritic cells there. At the ultrastructural level, close membrane contacts between bare axolemmal areas of unmyelinated nerve fibers and processes of MHC class II-expressing cells were observed. These results demonstrate close anatomical relationships of nerve fibers from various sources with antigen presenting dendritic cells in the visceral domain and suggest modulation of antigen presentation by the autonomic nervous system.