Risk factors for relapse of autoimmune hepatitis in Japan: A nationwide survey.

AIM: Some autoimmune hepatitis (AIH) patients experience relapse during their clinical course, and some risk factors for relapse have been identified previously using a relatively small sample size. The aim of the present study was to identify the risk factors for relapse in recently diagnosed AIH patients using a nationwide survey in Japan. METHODS: The nationwide survey performed in Japan in 2018 of AIH patients diagnosed between 2014 and 2017 was re-evaluated. A total of 614 patients who received corticosteroids were enrolled in the present study. Associations between relapse and patients' characteristics at diagnosis were evaluated using logistic regression analysis. RESULTS: Relapse was identified in 143 (23.3%) patients after remission. At the time of diagnosis of the disease, there were significant differences in the γ-glutamyl transpeptidase (γ-GTP) level, prevalence of liver cirrhosis, and degree of liver fibrosis. Multivariable logistic regression analysis showed that γ-GTP elevation and liver cirrhosis were significantly associated with relapse. CONCLUSION: The γ-GTP level at diagnosis could help identify AIH patients at higher risk of relapse.

Differences in autoimmune hepatitis based on inflammation localization.

Histopathology is essential for the diagnosis and evaluation of disease activity of autoimmune hepatitis (AIH). We aimed to elucidate the characteristics of AIH from the localization of inflammation. We re-evaluated a nationwide survey that was performed in Japan in 2018 of AIH patients diagnosed between 2014 and 2017. A total of 303 patients were enrolled, and the clinical and treatment characteristics were compared between the patients with predominantly portal inflammation (230 patients) or lobular inflammation (73 patients). AIH patients with lobular inflammation had a higher probability of being diagnosed with acute hepatitis than those with portal inflammation. Liver enzyme levels were higher in patients with lobular inflammation, whereas immunoglobulin G levels were higher in patients with portal inflammation. The prevalence of an alanine aminotransferase level < 30 U/L after 6 months of treatment was significantly higher in patients with lobular inflammation than in those with portal inflammation (81.7% vs. 67.3%, P = 0.046). The localization of inflammation may be useful for evaluating the onset of AIH.

IgM response is a prognostic biomarker of primary biliary cholangitis treated with ursodeoxycholic acid and bezafibrate.

BACKGROUND AND AIM: Primary biliary cholangitis (PBC) patients who are refractory to ursodeoxycholic acid (UDCA) are at risk for progression to cirrhosis and liver failure. Bezafibrate could be an alternative second-line therapeutic option in these patients. This study aimed to evaluate the long-term outcome(s) of combined UDCA and bezafibrate therapy in UDCA-refractory PBC patients and identify prognostic factors. METHODS: Among 445 patients treated with UDCA, 150 patients inadequately responded to UDCA monotherapy and received long-term UDCA plus bezafibrate (median, 15 years). Data from these patients were used for this retrospective analysis. RESULTS: Combination therapy resulted in significant improvements in serum biochemistry and liver transplantation risk estimated using the UK-PBC-risk and the GLOBE scores. The cumulative normalization rates of alkaline phosphatase, gamma-glutamyltransferase, and immunoglobulin M (IgM) were significantly higher in patients without cirrhosis-related symptoms or liver-related events than in those with them. Overall, IgM constantly emerged as a significant factor associated with cirrhosis-related symptoms and liver-related events at all time points. Cumulative survival rates were significantly lower in patients with IgM ≥ 240 mg/dL than in patients with IgM < 240 mg/dL. Thus, normalization of IgM levels was a good surrogate predictor of long-term prognosis. None of the patients discontinued combination therapy due to any adverse events during the follow-up period. CONCLUSIONS: Our findings point to the beneficial effects of long-term UDCA plus bezafibrate combination therapy for UDCA-refractory PBC patients, and IgM response can be a useful predictive biomarker of long-term clinical outcomes.

Genome-wide association studies identify PRKCB as a novel genetic susceptibility locus for primary biliary cholangitis in the Japanese population.

A previous genome-wide association study (GWAS) performed in 963 Japanese individuals (487 primary biliary cholangitis [PBC] cases and 476 healthy controls) identified TNFSF15 (rs4979462) and POU2AF1 (rs4938534) as strong susceptibility loci for PBC. In this study, we performed GWAS in additional 1,923 Japanese individuals (894 PBC cases and 1,029 healthy controls), and combined the results with the previous data. This GWAS, together with a subsequent replication study in an independent set of 7,024 Japanese individuals (512 PBC cases and 6,512 healthy controls), identified PRKCB (rs7404928) as a novel susceptibility locus for PBC (odds ratio [OR] = 1.26, P = 4.13 × 10-9). Furthermore, a primary functional variant of PRKCB (rs35015313) was identified by genotype imputation using a phased panel of 1,070 Japanese individuals from a prospective, general population cohort study and subsequent in vitro functional analyses. These results may lead to improved understanding of the disease pathways involved in PBC, forming a basis for prevention of PBC and development of novel therapeutics.

CD4+ T cells from patients with primary biliary cholangitis show T cell activation and differentially expressed T-cell receptor repertoires.

AIM: Primary biliary cholangitis (PBC) is an autoimmune liver disease with unknown pathogenesis. In PBC, activation of T-cell receptor (TCR) signaling is associated with inflammatory cytokine production through N-Ras upregulation. Although the CD4+ T cell TCR repertoire could be associated with PBC pathogenesis, it has not been evaluated. Thus, we analyzed the PBC-CD4+ T cell TCR repertoire using next generation sequencing (NGS). METHODS: Four PBC patients (one treatment-naïve and three receiving ursodeoxycholic acid) and three healthy individuals were enrolled. NRAS expression in CD4+ T cells was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). N-Ras dynamics in CD4+ T cells were assessed by qRT-PCR and GTP-N-Ras activation assay. The TCR α- (TRA) and ß-chain (TRB) repertoires on CD4+ T cells were analyzed by NGS and profiled using hierarchical analysis. Motif analysis was undertaken to elucidate the structure of PBC-specific TCRs. RESULTS: NRAS was upregulated in PBC relative to control CD4+ T cells (P < 0.05), and N-Ras enhanced T cell activation in CD4+ T cells. Among 2668 TRAs and 841 TRBs, 20 and 11, respectively, were differentially expressed in PBC compared to that in controls (P < 0.05, fold-change >2). Among them, TRAV29/J22, TRBV6-5/J2-6, and TRBV10-1/J2-1 were expressed in PBC but the expression was negligible in the controls, with more mature and longer forms observed in PBC-CD4+ T cells. CONCLUSIONS: N-Ras was upregulated in PBC-CD4+ T cells, and it enhanced TCR activation, indicating that PBC-CD4+ T cells were activated by N-Ras upregulation with differentially expressed TCR repertoires on their surfaces.

miR-425 regulates inflammatory cytokine production in CD4+ T cells via N-Ras upregulation in primary biliary cholangitis.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is an autoimmune liver disease of unknown pathogenesis. Consequently, therapeutic targets for PBC have yet to be identified. CD4+ T cells play a pivotal role in immunological dysfunction observed in PBC, and therefore, microRNA (miRNA) and mRNA expression were analysed in CD4+ T cells, to investigate PBC pathogenesis and identify novel therapeutic targets. METHODS: Integral miRNA and mRNA analysis of 14 PBC patients and ten healthy controls was carried out using microarray and quantitative real-time polymerase chain reaction (qRT-PCR), with gene set enrichment analysis. The functional analyses of miRNA were then assessed using reporter and miRNA-overexpression assays. RESULTS: The integral analysis of miRNA and mRNA identified four significantly downregulated miRNAs (miR-181a, -181b, -374b, and -425) related to the T cell receptor (TCR) signalling pathway in CD4+ T cells of PBC. N-Ras, a regulator of the TCR signalling pathway, was found to be targeted by all four identified miRNAs. In addition, in vitro assays confirmed that decreased miR-425 strongly induced inflammatory cytokines (interleukin [IL]-2 and interferon [IFN]-γ) via N-Ras upregulation in the TCR signalling pathway. CONCLUSION: The decreased expression of four miRNAs that dysregulate TCR signalling in PBC CD4+ T cells was identified. miR-425 was demonstrated as an inflammatory regulator of PBC via N-Ras upregulation. Therefore, the restoration of decreased miR-425 or the suppression of N-Ras may be a promising immunotherapeutic strategy against PBC. LAY SUMMARY: Primary biliary cholangitis (PBC) is an autoimmune liver disease, but the causes are unknown. MicroRNAs are molecules known to regulate biological signals. In this study, four microRNAs were identified as being decreased in PBC patients, leading to activation of T cell receptor signalling pathways, involved in inflammation. One particular target, N-Ras, could be an attractive and novel immunotherapeutic option for PBC. TRANSCRIPT PROFILING: Microarray data are deposited in GEO (GEO accession: GSE93172).

A Macrophage Migration Inhibitory Factor Polymorphism Is Associated with Autoimmune Hepatitis Severity in US and Japanese Patients.

BACKGROUND: The pathogenesis of autoimmune hepatitis (AIH) is incompletely understood. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine implicated in the pathophysiology of multiple autoimmune diseases. We recently reported that MIF expression was increased in a US AIH cohort. MIF expression in non-Western AIH patients is unknown. A MIF-173 GC single nucleotide polymorphism in the MIF promoter (rs755622) is clinically associated with steroid resistance in several inflammatory disorders but has not been evaluated in AIH. AIM: To compare MIF polymorphisms and their relationship to clinical parameters in AIH patients from the USA and Japan. METHODS: DNA and matched sera from AIH patients and healthy controls from Japan (N = 52) were compared to the US group. Serum concentrations of MIF and its circulating receptor CD74 were measured by ELISA. MIF-173 GC (rs755622) and MIF-794 CATT5-8 (rs5844572) polymorphisms were analyzed by standard methods. MIF genotypes were correlated with serum ALT and steroid requirements. RESULTS: Serum MIF was increased in Japanese AIH patients versus local controls, in agreement with the US AIH patients. Within both AIH groups, ALT was higher in CC/GC versus GG patients. Further, the steroid requirement was higher in AIH patients with GC/CC genotypes from both groups. In the Japanese patient group, the GC/CC genotype also was associated with acute symptomatic presentation. CONCLUSIONS: The MIF-173 CC/GC genotypes may be associated with both higher ALT and maintenance steroid requirements in AIH patients from the USA and Japan. This polymorphism could be a marker of disease severity in AIH patients.

Genome-wide association study identifies TNFSF15 and POU2AF1 as susceptibility loci for primary biliary cirrhosis in the Japanese population.

For the identification of susceptibility loci for primary biliary cirrhosis (PBC), a genome-wide association study (GWAS) was performed in 963 Japanese individuals (487 PBC cases and 476 healthy controls) and in a subsequent replication study that included 1,402 other Japanese individuals (787 cases and 615 controls). In addition to the most significant susceptibility region, human leukocyte antigen (HLA), we identified two significant susceptibility loci, TNFSF15 (rs4979462) and POU2AF1 (rs4938534) (combined odds ratio [OR] = 1.56, p = 2.84 × 10(-14) for rs4979462, and combined OR = 1.39, p = 2.38 × 10(-8) for rs4938534). Among 21 non-HLA susceptibility loci for PBC identified in GWASs of individuals of European descent, three loci (IL7R, IKZF3, and CD80) showed significant associations (combined p = 3.66 × 10(-8), 3.66 × 10(-9), and 3.04 × 10(-9), respectively) and STAT4 and NFKB1 loci showed suggestive association with PBC (combined p = 1.11 × 10(-6) and 1.42 × 10(-7), respectively) in the Japanese population. These observations indicated the existence of ethnic differences in genetic susceptibility loci to PBC and the importance of TNF signaling and B cell differentiation for the development of PBC in individuals of European descent and Japanese individuals.

Autoantibody status and histological variables influence biochemical response to treatment and long-term outcomes in Japanese patients with primary biliary cirrhosis.

AIM: The aim of the present study is to evaluate the factors influencing biochemical response to treatment and the value of biochemical response for predicting long-term outcomes in Japanese patients with primary biliary cirrhosis (PBC). METHODS: Biochemical response to ursodeoxycholic acid (UDCA) or UDCA plus bezafibrate was defined as good (≤upper limit of normal [ULN]), fair (≤1.5 × ULN) or poor (>1.5 × ULN) at 2 years after initiation of UDCA treatment. Associations between various factors (including age, sex, autoantibody status and histological variables at baseline), biochemical response to treatment and long-term outcomes were evaluated in 164 Japanese PBC patients. RESULTS: Anti-gp210 positivity and a higher bile duct loss score were significant risk factors for worse alkaline phosphatase (ALP) response (odds ratios [OR], 2.78 and 1.85, respectively). Age, anti-gp210 positivity and anticentromere positivity were significant risk factors for worse alanine aminotransferase (ALT) response (OR, 1.05, 4.0 and 2.77, respectively). Anti-gp210 positivity and a higher hepatitis score were significant risk factors for worse immunoglobulin (Ig)M response (OR, 2.10 and 2.06, respectively). Worse ALP and IgM response were significant risk factors for progression to late-stage disease without jaundice (OR, 2.27 and 2.32, respectively). Worse ALT response was a significant risk factor for progression to late-stage disease with persistent jaundice (OR, 11.11). CONCLUSION: Biochemical response to treatment at 2 years, which is influenced by autoantibody status and histological variables at baseline, can predict long-term outcomes in Japanese patients with PBC.

[Hepatitis C virus infection and hemodialysis: update 2014].

Since hepatitis C virus (HCV) infection is much common among hemodialysis (HD) patients, the natural history of HCV is not sufficiently understood. It has been reported that HCV infection is one of the major risk factors for the lower survival among HD patients. Recent progression of the therapy involving direct acting antiviral (DAA) can eliminate HCV infection in almost HCV infected patients, resulting HD patients should also get this benefit. Though HD patients have many restricted factors for accepting anti HCV therapy, the significance of clinical usefulness of interferon beta that has less adverse effect should be more deeply considered with newly developed DAAs for eliminating HCV infected HD patients.

Sal-like protein 4 (SALL4), a stem cell biomarker in liver cancers.

UNLABELLED: Liver cancers, including hepatocellular carcinomas (HCCs), cholangiocarcinomas (CCs), and fibrolamellar HCCs (FL-HCCs) are among the most common cancers worldwide and are associated with a poor prognosis. Investigations of genes important in liver cancers have focused on Sal-like protein 4 (SALL4), a member of a family of zinc finger transcription factors. It is a regulator of embryogenesis, organogenesis, pluripotency, can elicit reprogramming of somatic cells, and is a marker of stem cells. We found it expressed in normal murine hepatoblasts, normal human hepatic stem cells, hepatoblasts and biliary tree stem cells, but not in mature parenchymal cells of liver or biliary tree. It was strongly expressed in surgical specimens of human HCCs, CCs, a combined hepatocellular and cholangiocarcinoma, a FL-HCC, and in derivative, transplantable tumor lines in immune-compromised hosts. Bioinformatics analyses indicated that elevated expression of SALL4 in tumors is associated with poor survival of HCC patients. Experimental manipulation of SALL4's expression results in changes in proliferation versus differentiation in human HCC cell lines in vitro and in vivo in immune-compromised hosts. Virus-mediated gene transfer of SALL4 was used for gain- and loss-of-function analyses in the cell lines. Significant growth inhibition in vitro and in vivo, accompanied by an increase in differentiation occurred with down-regulation of SALL4. Overexpression of SALL4 resulted in increased cell proliferation in vitro, correlating with an increase in expression of cytokeratin19 (CK19), epithelial cell adhesion molecules (EpCAM), and adenosine triphosphate (ATP)-binding cassette-G2 (ABCG2). CONCLUSION: SALL4's expression is an indicator of stem cells, a prognostic marker in liver cancers, correlates with cell and tumor growth, with resistance to 5-FU, and its suppression results in differentiation and slowed tumor growth. SALL4 is a novel therapeutic target for liver cancers.

Usefulness of combined application of double-filtration plasmapheresis and twice-daily injections of interferon-ß in hemodialysis patients with hepatitis C virus genotype 1b infection and a high viral load.

Hepatitis C virus (HCV) infection is common among hemodialysis (HD) patients and has been recognized as an important prognostic factor. Therefore, the aggressive antiviral therapy is necessary for HCV infection in HD patients. However, various treatment limitations exist in HD patients such as the inability to use ribavirin. We have previously reported that HCV RNA can be eradicated by administration of interferon (IFN)-ß during HD in patients with HCV infection caused by genotypes known to be sensitive to IFN therapy and low serum HCV RNA levels. In this case report, we tried to clarify the efficacy of combined application of double-filtration plasmapheresis (DFPP) and IFN-ß in HD patients with HCV genotype 1b infection and high serum HCV RNA levels. We report two HD patients with HCV genotype 1b infection and high viral loads who were successfully treated by five sessions of DFPP undertaken prior to treatment with IFN-ß (twice-daily injections for 2 weeks). HCV was eradicated by this combination therapy in both patients. We revealed the efficacy of combined application of DFPP and IFN-ß in HD patients with HCV genotype 1b infection and high serum HCV RNA levels. This combined therapy may be useful for the HD patients who are resistant to conventional IFN monotherapy.

Multicenter validation study of anti-programmed cell death-1 antibody as a serological marker for type 1 autoimmune hepatitis.

AIM: Recently, serum levels of anti-programmed cell death-1 (anti-PD-1) antibodies have been reported to be useful for the discrimination of type 1 autoimmune hepatitis (AIH) from drug-induced liver injury (DILI) and to be associated with clinical features of type 1 AIH. This multicenter study aimed to validate the usefulness of serum anti-PD-1 antibody as a serological marker for type 1 AIH. METHODS: Serum samples before the initiation of corticosteroid treatment were obtained from 71 type 1 AIH patients and 37 DILI patients. Serum levels of anti-PD-1 antibodies were measured by indirect enzyme-linked immunosorbent assay. RESULTS: Serum levels of anti-PD-1 antibodies were higher in type 1 AIH patients than in DILI patients (P < 0.001). The receiver-operator curve analysis showed that serum levels of anti-PD-1 antibodies were useful for the discrimination of type 1 AIH from DILI (area under the curve, 0.80). On the other hand, the multivariate Cox proportional hazard model showed that positivity for serum anti-PD-1 antibody, probable diagnosis based on the revised scoring system proposed by the International Autoimmune Hepatitis Group, and prothrombin activity of less than 60% were associated with the later normalization of serum transaminase levels. During the clinical course, the disease relapsed more frequently in patients positive for serum anti-PD-1 antibody (36% vs 11%). CONCLUSION: This study suggests that serum anti-PD-1 antibody is useful for the diagnosis of type 1 AIH as an auxiliary diagnostic marker, and that serum levels of anti-PD-1 antibodies reflect clinical features of type 1 AIH.

Autoimmune Hepatitis with Metabolic Dysfunction-associated Fatty Liver Disease.

Objective Metabolic-associated fatty liver disease (MAFLD) has only recently been proposed; therefore, the characteristics of patients with autoimmune hepatitis (AIH) and MAFLD remain unclear. This study evaluated the effect of MAFLD on AIH patients with AIH. Methods We reevaluated the Japanese Nationwide Survey of AIH in 2018, which involved a survey of patients diagnosed with AIH between 2014 and 2017. We categorized patients with AIH according to the presence or absence of MAFLD and compared the clinical characteristics between the two groups. Results A total of 427 patients (77 men and 350 women) were included in this study. The overall prevalence of MAFLD was 10.5%. Compared to AIH patients without MAFLD, AIH patients with MAFLD had the following characteristics at the time of the AIH diagnosis: (1) a higher body mass index, (2) a higher prevalence of hypertension, (3) mild elevation of hepatobiliary enzymes and total bilirubin, and (4) histologically progressive fibrosis. However, the levels of hepatobiliary enzymes and total bilirubin after treatment were significantly higher in AIH patients with MAFLD than in those without MAFLD. Conclusions AIH patients with MAFLD had characteristics different from those of AIH patients without MAFLD. These findings could help increase our understanding of patients with AIH with MAFLD.

Application and validation of a new histologic staging and grading system for primary biliary cirrhosis.

BACKGROUND: We proposed a new grading and staging system for primary biliary cirrhosis (PBC), which takes into account the degree of both chronic cholangitis activity (CA) and hepatitic activity (HA) for grading disease activity and that of fibrosis, bile duct loss, and chronic cholestasis for staging. In this study, we validated our new system. METHODS: Using liver biopsy specimens from 166 cases of PBC, chronic cholangitis with mild periductal lymphoplasmacytic infiltration, including chronic nonsuppurative destructive cholangitis, and the combined activity of interface hepatitis and lobular hepatitis were categorized into 4 grades on the basis of their degree and distribution (CA0-3 and HA0-3, respectively). For staging, because orcein staining was not available in this study, 2 criteria (fibrosis and bile duct loss) were independently scored from 0 to 3 on the basis of their degrees, and a final stage score was created from the sum. RESULTS: Although there was a relatively uniform distribution of CA0/1/2/3 cases, the cases of HA0/1/2/3 were distributed as 21%, 64%, 13%, and 3%, respectively, with a prominent number of cases categorized as having none or mild HA. The distribution of stages 1 to 4 using our system was considerably different from that using the classic system and, importantly, showed a correlation with patient outcome. CONCLUSIONS: Our system revealed that the activities of chronic cholangitis and hepatitis did not correlate with each other in terms of degree and that our staging system properly reflected the outcome of PBC patients. The present study could validate the effectiveness of this new system for characterizing the pathologic condition of PBC.

Prognosis of autoimmune hepatitis showing acute presentation.

AIM: The number of patients with autoimmune hepatitis (AIH) showing acute presentation has increased. This study aimed to assess their prognosis. METHODS: A survey of AIH patients by sending questionnaires was performed, and 96 patients showing acute presentation were investigated. RESULTS: The median age was 58 years and 78 patients (81%) were female. Eighty-four patients (88%) were positive for antinuclear antibody and/or anti-smooth muscle antibody. The median serum immunoglobulin G level was 2252 mg/dL. Twenty-five patients (26%) showed histological acute hepatitis. As initial treatment, 88 patients (92%) were treated with corticosteroid, and 28 of them received pulse steroid treatment. Overall, 11 patients (11%) reached fatal outcomes (nine death and two liver transplantation). Patients with histological acute hepatitis showed higher serum bilirubin levels, lower prothrombin activities and higher prothrombin time-international normalized ratios (PT-INR) and reached fatal outcomes more frequently. With a multivariate logistic regression analysis, prothrombin activity and PT-INR at presentation was associated with fatal outcomes. Nine of 13 patients (69%) showing prothrombin activity of 40% or lower at presentation and nine of 19 patients (47%) showing PT-INR of 1.5 or higher reached fatal outcomes. Furthermore, of 13 patients showing prothrombin activity of 40% or lower and/or PT-INR of 1.5 or higher at presentation who were treated with pulse steroid treatment, four (31%) died from infectious disease. CONCLUSION: Prothrombin activity and PT-INR are prognostic factors for AIH showing acute presentation. Physicians should pay attention to the development of infectious disease when pulse steroid treatment is performed.

Changes in lifestyle-related behaviour during the COVID-19 pandemic in Japan: a questionnaire survey for examinees who underwent an annual health check-up.

OBJECTIVE: To investigate the effect of the coronavirus disease (COVID-19) pandemic on lifestyle behaviour and clinical data in a population who underwent an annual health check-up in Tokyo, Japan. METHODS: A self-report questionnaire was completed regarding changes in their physical activities, diet, alcohol intake, smoking and mental stress. For those recommended to undergo further examination or treatment, their intention to do so was also questioned. The clinical results of the check-ups across three different periods (before and during the pandemic and survey period) were statistically compared. RESULTS: During the survey period, 838 examinees responded. While physical activities decreased due to teleworking, changes in food intake and dietary patterns were varied. Furthermore, changes in mental stress were also diverse. As for the intention to undergo further clinical examination or treatment, 23.5% answered that they thought they would wait until the government lifted the state of emergency or the pandemic subsided. Compared with before the pandemic, diastolic blood pressure, liver function, kidney function and bone density tended to deteriorate. CONCLUSIONS: The COVID-19 pandemic affected the lifestyle of the current study population. To prepare for future outbreaks, real-world information should be collected and shared so that effective measures for health promotion can be developed.

Accumulation of functional regulatory T cells in actively inflamed liver in mouse dendritic cell-based autoimmune hepatic inflammation.

Participation of Tregs in the generation of autoimmune hepatic inflammation (AHI) was examined using a newly established dendritic cell (DC)-based mouse model of hepatitis. The inflammatory activity of AHI peaked 21 days after DC vaccination. Forkhead box P3 (Foxp3) expression on day 21 was significantly increased in the liver but was decreased in the spleen. CD4(+)CD25(+) Tregs from the liver on day 21 showed inhibitory activity against the proliferation of CD4(+)CD25(-) T cells. On day 21, the expression of CXCR3 on Tregs and its ligand CXCL9 in hepatic tissue was upregulated, and levels of mRNA of transforming growth factor (TGF)-beta and IL-2, essential for Treg differentiation, in the liver were also increased. Suppression of AHI activity by prednisolone treatment decreased Treg accumulation in the liver. Accumulation of Tregs might occur through Treg recruitment mediated by CXCR3/CXCL9 interaction and expansion in the liver by upregulated TGF-beta and IL-2.

Sall4 regulates cell fate decision in fetal hepatic stem/progenitor cells.

BACKGROUND & AIMS: Fetal hepatic stem/progenitor cells, called hepatoblasts, differentiate into both hepatocytes and cholangiocytes. The molecular mechanisms regulating this lineage segmentation process remain unknown. Sall4 has been shown to be among the regulators of organogenesis, embryogenesis, maintenance of pluripotency, and early embryonic cell fate decisions in embryonic stem cells. The expression and functional roles of Sall4 during liver development have not been elucidated. We here provide their first description in hepatoblasts. METHODS: To investigate functions of Sall4 in fetal liver development, Dlk(+)CD45(-)Ter119(-) hepatoblasts derived from embryonic day 14 mouse livers were purified, and in vitro gain and loss of function analyses and in vivo transplantation analyses were performed using retrovirus- or lentivirus-mediated gene transfer. RESULTS: We demonstrated that Sall4 was expressed in fetal hepatoblasts but not adult hepatocytes. The expression level of Sall4 gradually fell during liver development. Overexpression of Sall4 in hepatoblasts significantly inhibited maturation induced by oncostatin M and extracellular matrix in vitro, as evidenced by morphologic changes and suppression of hepatic maturation marker gene expression. When bile duct-like structures were induced by collagen gel-embedded culture, overexpression of Sall4 markedly augmented size and number of cytokeratin19(+)-branching structures. Knockdown of Sall4 inhibited formation of these branching structures. With in vivo transplantation, Sall4 enhanced differentiation of cytokeratin19(+)-bile ducts derived from transplanted hepatoblasts. CONCLUSIONS: These results suggest that Sall4 plays a crucial role in controlling the lineage commitment of hepatoblasts not only inhibiting their differentiation into hepatocytes but also driving their differentiation toward cholangiocytes.